Antibodies specific for a cell surface molecule mediating cell adhesion and signal transmission, cells secreting such antibodies, and methods of making and using such antibodies

ABSTRACT

Novel cell surface molecules recognized by monoclonal antibodies against a cell surface molecule of lymphocytic cells that play an important role in autoimmune diseases and allergic diseases have been isolated, identified, and analyzed for their functions. The cell surface molecules are expressed specifically in thymocytes, lymphocytes activated by ConA-stimulation, and peripheral blood lymphocytes, and induce cell adhesion. Antibodies against the cell surface molecules significantly ameliorate pathological conditions of autoimmune diseases and allergic diseases.

TECHNICAL FIELD

[0001] The present invention relates to novel cell surface molecules ofmammals; polypeptides and their fragments constituting the molecules;fusion polypeptides comprising the polypeptide fragments andimmunoglobulin fragments; genes encoding the polypeptides and thefragments; vectors comprising the genes; transformants into which thevectors are introduced; antibodies having reactivity to the polypeptidesor cell surface molecules comprising the polypeptides; hybridomasproducing the antibodies; pharmaceutical compositions comprising thepolypeptide fragments or the fusion polypeptides; pharmaceuticalcompositions comprising the antibodies; transgenic mice; and knockoutmice.

BACKGROUND ART

[0002] A living body of mammals has immune response systems thatexcludes pathogenic microorganisms (viruses, bacteria, parasites, etc.)or foreign bodies (both are called “antigen” in the following) that haveinvaded the living body. One of them is called natural immune responsesystem, another acquired immune response system. The former is anexclusion mechanism comprising phagocytosis by phagocytes(polymorphonuclear leukocytes, monocytes, macrophages, etc.), attack bynatural killer (NK) cells, and non-specific recognition such asopsonization of antigen by complements. The latter, acquired immuneresponse system, is an exclusion mechanism by lymphocytes (mainly, Tcells and B cells) that acquired the specificity to the antigen (namely,activated lymphocytes). B cells that acquired antigen specificityattacks the antigen existing outside of the cells through production ofantibodies specific to the antigen. T cells that acquired antigenspecificity (namely, activated T cells) are classified into helper Tcells and cytotoxic T cells (cytotoxic lymphocyte, CTL). The helper Tcells regulate a differentiation of B cells and a production ofantibodies, and destroy the antigen cooperating with phagocytes. Thelatter, CTLs attack virus-infected cells and so on by themselves(Experimental Medicine: SUPPLEMENT, “Bio Science Term Library,Immunity”, Yodosha, pp. 14-17 (1995)).

[0003] This acquisition of antigen specificity by T cells (namely,activation of T cells) is initiated through recognition by T cells theantigen presented by antigen-presenting cells (APC) such as macrophage,B cells, or dendritic cells. Antigen-presenting cells process theantigens so incorporated and present these processed antigens throughbinding them to major histocompatibility complex (MHC). T cells receivesprimary signal for activation of the cells (or acquisition ofspecificity) by recognizing the processed antigens presented byantigen-presenting cells through a complex between T cell receptor (TcR)and CD3 antigen existing on the surface of the cell membrane (TcR/CD3complex).

[0004] However, the TcR/CD3 complex-mediated primary signal alone cannotactivate T cells sufficiently and leads to unresponsiveness or clonalanergy, so that the cells can not react with any stimulation receivedthereafter. The autocrine of interleukin 2 (IL-2) is necessary for Tcells to be activated, to be differentiated into antigen specific T cellclones, and to be proliferated. In clonal anergy, T cells areinactivated due to no production of IL-2 and no cell division. Namely,the activation of T cells accompanied by production of cytokines such asIL-2 requires the secondary signal following the first signal throughTcR/CD3 complex. This secondary signal is called costimulatory signal.

[0005] T cells receive this secondary signal and transmit it into thecells by interacting (cell adhesion) with molecules other than MHC onantigen-presenting cells through other molecules other than TcR/CD3complex on the T cell surface. This secondary signal avoids cell anergy(clonal anergy) and activates the cells.

[0006] Although some part of the mechanism of the secondary signaltransmission between antigen-presenting cells and lymphocytes such as Tcells have not yet been elucidated in detail, studies so far haverevealed that an important factor for the secondary signal transmissionis the interaction of CD28 (also named Tp44, T44, or 9.3 antigen), whichis a cell surface molecule expressed mainly on T cells and thymus cells,with CD80 (also named B7-1, B7, BB1, or B7/BB1), which is a cell surfacemolecule expressed on antigen-presenting cells (macrophages, monocytes,dendritic cells, and so on etc.) and with CD86 (also named B7-2 or B70),which is also a cell surface molecule on antigen-presenting cells(namely, cell adhesion through the binding between these molecules).Moreover, it has been experimentally elucidated that the interaction ofCytolytic T lymphocyte associated antigen 4 (CTLA-4), whose expressionis thought to be enhanced depending on the secondary signal, with theCD80 (B7-1) and CD86 (B7-2) (namely, cell adhesion through the bindingbetween these molecules) also plays an important role in the regulationof T cell activation by the secondary signal. In other words, theregulation of T cell activation by the transmission of the secondarysignal involves, at least the interaction between CD28 and CD80/CD86,the enhancement of CTLA-4 expression, which is thought to depend on theinteraction, and the interaction between CTLA-4 and CD80/CD86.

[0007] CD28 is known to be a costimulator molecule transmitting thesecondary signal (costimulatory signal) required for the activation of Tcells and for the avoidance of anergy. The secondary signal transmittedby binding this molecule to costimulator molecules, CD80 (B7-1) and CD86(B7-2), on antigen-presenting cells (namely, cell adhesion through thebinding between these molecules), stabilizes mRNA of Th1-type cytokinesand consequently promotes production by T cells of a large amount ofproduction of Th1-type cytokines such as I1-2, IFNγ, and TNFα. Theexpression of CTLA-4 is induced by the primary signal transmittedthrough TcR/CD3, and the expression is also enhanced by the secondarysignal transmitted by the binding between CD28 and CD80. It is beingrevealed that CTLA-4 receives these signals to work to inhibit T cellfunction, which is contrary to the activation of T cells by thesecondary signal transmitted by CD28.

[0008] Human CD28 and CTLA-4 are I-type glycoproteins whose molecularweights are 44 kD and 41 to 43 kD, respectively. Both have animmunoglobulin-like domain, belong to the immunoglobulin superfamily,and have both function as a cell adhesion molecule and function as asignal transmission molecule.

[0009] Human CD28 forms a homodimer with a disulfide bond while CTLA-4exists as a monomer. Both CD28 and CTLA-4 genes are located at “2q33” onhuman chromosome and “1C” on mouse chromosome, and are composed of four(4) exons. Human CD28 and CTLA-4 are composed of 220 and 223 aminoacids, respectively, including the leader sequences, and amino acidhomology between them is 20 to 30%.

[0010] The ligands for CD28 and CTLA-4 are CD80 (B7-1) and CD86 (B7-2)in human and mice. CTLA-4 has about 20 times as higher affinity to bothligands as CD28. It has been elucidated that the amino acid sequencestructures “MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)” conserved through animalspecies is important for the binding of CD28 and CTLA-4 to CD80 (B7-1).It has also been reported that, when CD28 is stimulated, PI3 kinase(phosphoinositide 3 kinase, PI3K) associates with the phosphorylatedtyrosine residue in a partial sequence “YMNM (Tyr-Met-Asn-Met)” of CD28“YMNM (Tyr-Met-Asn-Met)” and that CD28 plays an important role inintracellular signal transmission through this “YxxM” structure.Furthermore, it has been reported that CTLA-4 also has a sequencerepresented by “YxxM,” namely “YVKM (Tyr-Val-Lys-Met)” in itscytoplasmic region and that, after being stimulated, SYP associates withthis sequence.

[0011] CD28 is expressed specifically in thymocytes and peripheral bloodT cells, and CTLA-4 is expressed specifically in activated T cells (CellEngineering: SUPPLEMENT, “Handbook of Adhesion Molecule”, Shujunsha, pp.93-102 (1994); ibid. pp. 120-136; Experimental Medicine: SUPPLEMENT,“BIO SCIENCE Term Library, Immunity”, Yodosha, pp. 94-98 (1995);Experimental Medicine: SUPPLEMENT, “BIO SCIENCE Term Library,Intracellular Signal Transduction”, Yodosha, pp. 58-59 (1997); NihonRinsho, Vol.55, No.6, pp. 215-220 (1997)).

[0012] In the regulation of T cell function (the activation and theinhibition of function of T cells), the importance of interactions amongmultiple molecules such as costimulator molecules (CD28, CD80 (B7-1),CD86 (B7-2), etc.) and CTLA-4, which cooperates with them, (in otherwords, cell adhesion through the binding between these molecules) hasthus been recognized, and this has been drawn attention to therelationship between these molecules and diseases, and the treatment ofdiseases by regulating the function of these molecules have been noted.

[0013] As described above, although a living body activates its acquiredimmune response system against antigens that are foreign bodies to theliving body (self), it also has immunological tolerance so as to show noimmune response against its own component (autoantigen). Ifimmunological tolerance breaks down by some reason, immune response tothe autoantigen occurs, autoantigen-reactive T cells are induced by thesame mechanism as mentioned above to fall into abnormal state ofimmunity, and various autoimmune diseases are caused.

[0014] In other words, since non-stimulated antigen presenting cells(APC) in normal tissues do not express costimulatory molecules when theimmune system of a living body is normal, T cells fall are in theunresponsiveness state to maintain immunological tolerance even ifautoantigen-reactive T cells, which reacts with autoantigen, exist. Ithas been suggested that in abnormal state of immunity, moreautoantigen-reactive T cells are activated due to abnormal excess andcontinuous expression of costimulatory molecules to thereby causeautoimmune diseases.

[0015] From such viewpoints recently, many attempts to treat for variousautoimmune diseases by modulating the transmission of costimulatorysignals, for example, the above-mentioned signal transmission betweenCD28/CTLA-4 and CD80/CD86, are proposed.

[0016] It has been observed CD80, a costimulatory molecule as the ligandof CD28 and CTLA-4, is abnormally expressed in the antigen presentingcells at the nidus of autoimmune disease such as rheumatoid arthritis,multiple sclerosis, autoimmune thyroiditis, allergic contact-typedermatitis, and chronic inflammatory dermatosis such as squamous lichenplanus, and psoriasis. From such observation, many attempts to treatvarious autoimmune diseases by modulating the function of CD80 have beenmade.

[0017] It has been proposed to block the function of CD80, by methodsusing an antibody against CD80, solubilized protein of CD28 that is aligand of CD80, and solubilized protein of CTLA-4 that is also a ligandof CD80. Particularly, based on the fact that the binding affinity ofCTLA-4 to CD80 is 20 or more times higher than that of CD28, therapeuticattempts using “solubilized CTLA-4,” specifically, the fusion protein(CTLA-4-IgFc) comprising the extracellular domain of “CTLA-4” and the Fcregion of human immunoglobulin IgG1, were performed in animal model andclinical tests (Nihon Rinsho, Vol. 55, No. 6, pp. 215-220 (1997)).

[0018] As shown in 1 to 5 below, therapeutic effects of CTLA-4-IgFc inmodel animals of autoimmune diseases has been reported.

[0019] 1. In a (NZB/NZW)F1 mouse, that is a model for human systemiclupus erythematosus (SLE), the production of autoantibodies and theonset of lupus nephritis were suppressed by administration ofCTLA-4-IgFc before the onset, and the pathologic conditions wereimproved by administration of the drug even after the onset (Science,Vol. 125, p. 1225-1227 (1994)).

[0020] 2. In experimental allergic encephalomyelitis (EAE), that is amodel for multiple sclerosis (MS), the onset was prevented by short-termadministration of CTLA-4-IgFc immediately after immunization (J. Clin.Invest., Vol.95, pp. 2783-2789 (1995)).

[0021] 3. In an NOD (non-obese diabetes) mouse, which is a model forinsulin dependent diabetes mellitus (IDDM), the onset rate wasremarkably decreased by administering CTLA-4-IgFc to the 2- or3-week-old female mouse for two weeks (J. Exp. Med. 181:1145-1155,1995).

[0022] 4. In rat nephritis by renal glomerulus basement membraneimmunity, Goodpasture's nephritis model, the improvement of the symptomhas been improved by the administration of CTLA-4-IgFc (Eur. J. Immunol.24:1249-1254, 1994).

[0023] 5. In type II collagen-induced arthritis (CIA) using a DBA/1mouse, that is a model for human rheumatoid arthritis, the onset ofarthritis was suppressed by the administering the test drug at the timeof immunization and the production of autoantibodies (IgG1 and IgG2)against collagen was inhibited (Eur. J. Immunol. 26:2320-2328, 1996).

[0024] The results of the experiments as mentioned above are have notyet clarified in detail the mechanism of the T cell activation byinteraction between costimulatory molecules and the related molecules(in other words, cell adhesion through the binding between thesemolecules). Other unknown molecules may be involved in this mechanism.

DISCLOSURE OF THE INVENTION

[0025] Pharmaceuticals useful for treating or preventing variousdiseases such as the above-mentioned autoimmune diseases, allergicdiseases, and inflammatory diseases can be developed if the mechanism ofthe activation of lymphocytes such as T cells by cell adhesion throughthe binding between molecules involved in the transmission of thesecondary signal essential for the activation of lymphocytes such as Tcells mentioned above and the mechanism of the regulation of lymphocytefunction are clarified, and known or unknown molecules capable ofmediating cell adhesion involved in the mechanism and of transmittingsignals are identified and characterized.

[0026] An objective of the present invention is to identify novel cellsurface molecules having both functions of mediating such cell adhesionand signal transmission, and to clarify its structural and biologicalcharacteristics. Another objective of the present invention is toprovide pharmaceuticals useful for treating or preventing variousautoimmune diseases and inflammatory diseases by using the novelmolecules or antibodies against the molecules.

[0027] In order to identify such useful molecules, the present inventorsfocused on the fact that lymphocytes such as T cells play an importantrole in autoimmune diseases, and the fact that cell adhesion areessential for the signal transmission of the secondary signal(costimulatory signal) from antigen presenting cells into lymphocytes,and planned to isolate and identify cell surface molecules that areexpressed specifically on lymphocytic cells and that mediate celladhesion.

[0028] The present inventors obtained monoclonal antibodies againstvarious cell surface molecules expressed on the surface of lymphocyticcells by immunizing animals against the lymphocytic cells, and isolatedand identified desired cell surface molecules that mediate cell adhesionusing the monoclonal antibodies so obtained. The methods used aredescribed in detail below.

[0029] The present inventors first administered rat lymphocytic cellline as an antigen to mice and prepared various monoclonal antibodies.Then, the monoclonal antibodies obtained were reacted with ratlymphocytic cells used as an antigen and tested the effect of themonoclonal antibodies given to the cells. As a result, one of themonoclonal antibodies was obtained has been found to agglutinate the ratlymphocytic cells strongly (this monoclonal antibody was designated“JTT-1 antibody”). Moreover, other one of the monoclonal antibodies wasfound to strongly inhibit the agglutination of rat lymphocytic cellsinduced by the “JTT-1 antibody” (this monoclonal antibody was designated“JTT.2 antibody”).

[0030] Since the agglutination of rat lymphocytic cells by “JTT-1antibody” was not inhibited by antibodies against Intercellular adhesionmolecule-1 (ICAM-1) or Lymphocyte function-associated antigen-1 (LFA-1),which are the most representative known cell adhesion moleculesexpressed on the cells, the present inventors thought that thisagglutination was caused by cell adhesion through unknown adhesionmolecules having that mediate cell adhesion.

[0031] Cell surface molecules (designated “JTT-1 antigen” and “JTT.2antigen”) recognized by each of these two monoclonal antibodies werethen identified, isolated, and characterized.

[0032] First, the analysis of the expression patterns of “JTT-1 antigen”and “JTT.2 antigen” in various cells were analyzed by flow cytometrybased on fluorescent antibody technique using “JTT-1 antibody” and“JTT-2 antibody.” While both “JTT-1 antigen” and “JTT.2 antigen” werestrongly expressed in activated lymphoblast cells (activated Tlymphoblast cells, activated B lymphoblast cells, etc.) activated bystimulating thymocytes and spleen cells with Concanavalin A (ConA), amitogen, in particular, in the activated lymphoblast cells, theexpression was hardly found in spleen cells not stimulated at all (thesecells are sometimes called “resting lymphocytes” herein). The expressionpatterns of molecules recognized by each of “JTT-1 antibody” and “JTT-2antibody” were almost the same.

[0033] Using an affinity column prepared by binding “JTT-1 antibody” toadsorbents, molecules trapped by the “JTT-1 antibody”, namely, “JTT-1antigens” were purified from the mixture of soluble cell surfacemolecules prepared from the above-described rat lymphocytic cells. Themolecular weights of these purified “JTT-1 antigens” were analyzed byimmunoprecipitation using “JTT-1 antibody” and “JTT-2 antibody” and bySDS-PAGE. As a result, it was found that molecules immunoprecipitated byeach of “JTT-1 antibody” and “JTT-2 antibody” were the same, and thateach molecule was a homodimer having different sugar chains.Specifically, when N-linked sugar chains were not digested, themolecules were identified as one molecule with about 47 kD undernon-reduction condition, and as two molecules with about 24 kD and about28 kD under reduction condition; and when N-linked sugar chains weredigested, the molecules were identified as one molecule with about 36 kDunder non-reduction condition and as one molecule with about 20 kD underreduction condition.

[0034] The adhesion of rat thymocytes to the plate coated by thepurified “JTT-1 antigen” was then analyzed. As a result, thymocytessignificantly adhered to the plate (namely, to “JTT-1 antigen”) only inthe presence of “JTT-1 antibody” and that the adhesion was significantlyinhibited in the co-presence of “JTT.2 antibody”, indicating that “JTT-1antigen” was the cell surface molecule mediating cell adhesion.

[0035] Next, the present inventors cloned genes encoding “JTT-1 antigen”from rat, human, and mouse, and analyzed their structures.

[0036] First, the cDNA encoding the full length of “rat JTT-1 antigen”was isolated from the cDNA library made from the lymphoblasts derivedfrom ConA-stimulated rat spleen by expression cloning method utilizingpanning method using “JTT-1 antibody” and a completely novel rat genewas isolated and identified by determining its nucleotide sequence bydideoxy method. The cDNA encoding the full length of “human JTT-1antigen” was also isolated from the cDNA library made fromConA-stimulated human peripheral blood lymphoblasts by plaquehybridization with using the cDNA encoding “rat JTT-1 antigen” soobtained as a probe and a completely novel human gene was isolated andidentified by determining its nucleotide sequence by dideoxy method.Similarly, the cDNA encoding the full length of “mouse JTT-1 antigen”was isolated from the cDNA library made from the lymphoblasts derivedfrom ConA-stimulated mouse spleen and a completely novel mouse gene wasisolated and identified by determining its nucleotide sequence bydideoxy method. Furthermore, the cDNA encoding the full length ofalternative splicing variant of “rat JTT-1 antigen” mentioned above wasisolated similarly from the cDNA library made from the rat thymoma cellline and another completely novel rat gene was isolated and identifiedby determining its nucleotide sequence by dideoxy method.

[0037] “JTT-1 antigen” was found to be a transmembrane protein (cellsurface molecule) composed of a signal sequence, an extracellular regionhaving the glycosylation site(s), a transmembrane region, and anintracellular region by hydropathy plot analysis of the amino acidsequence encoded by the isolated cDNA of “human JTT-1 antigen”. Homologysearch with known molecules revealed that of “JTT-1 antigens” from rat,human, and mouse had no significant homology to any known moleculesincluding cell adhesion molecules, indicating that they are novel cellsurface molecules that mediates cell adhesion.

[0038] As the result that of motif search based on the amino acidsequence of “human JTT-1 antigen”, it was found that “human JTT-1antigen” had structural similarity with the above-mentioned “CD28”, acell surface molecule on lymphocytes such as T cells, which transmitscostimulatory signal important for T cell activation through celladhesion and with “CTLA-4”, a cell surface molecule on lymphocytes suchas T cells, which regulates the functions of activated lymphocytes suchas activated T cells, cooperating with the signal.

[0039] The structural similarity is as follows.

[0040] 1. 20 or more amino acid residues including cysteine residues arehighly conserved.

[0041] 2. Proline repeating sequence “Pro-Pro-Pro (PPP)” essential asthe ligand binding region, is conserved in the extracellular region.

[0042] 3. A sequence “Tyr-Xaa-Xaa-Met (YxxM)” (Xaa and x represents anyamino acid) sequence essential as the signal transmitting region isconserved in the cytoplasmic region.

[0043] The locus of the gene encoding “mouse JTT-1 antigen” on mousechromosome was found to be “1C3”, which is the same location as that ofmouse “CD28” and “CTLA-4” using fluorescence in situ hybridization(FISH) method.

[0044] Next, the effectiveness of therapy of autoimmune diseases andallergic diseases by regulating the function of “JTT-1 antigen”, wasexamined by experiments in which “JTT-2 antibody” mentioned above wasadministered to model rats for experimental allergic encephalomyelitis(EAE) and glomerulus basement membrane (GBM) nephritis. It was foundthat the pathological states were significantly suppressed in bothdisease model animals, and that autoimmune diseases or allergic diseasescan be treated by regulating the functions of “JTT-1 antigen”.

[0045] It was also found that the monoclonal antibody against “humanJTT-1 antigen” significantly proliferated human peripheral bloodlymphocytes, and that the proliferation was further enhanced in theco-presence of a monoclonal antibody against CD3 constituting a TcR/CD3complex on T cells, which receives the primary signal essential for Tcell activation from antigen presenting cells, indicating that “JTT-1antigen” was a cell surface molecule involved in signal transmissioninto lymphocytes.

[0046] Furthermore, the present inventors succeeded in producing afusion polypeptide comprising of the extracellular region of “humanJTT-1 antigen” and Fc region of human immunoglobulin. The fusionpolypeptide is useful as pharmaceuticals for treating autoimmunediseases, allergic diseases, and inflammatory diseases by regulating the“JTT-1 antigen” and/or its ligand.

[0047] Moreover, the present inventors succeeded in preparing atransgenic mouse into which a gene encoding “JTT-1 antigen” of otheranimal species was introduced. The transgenic mouse is useful foranalyzing detailed functions of “JTT-1 antigen” and for developingpharmaceuticals for treating autoimmune diseases, allergic diseases, andinflammatory diseases. The inventors also produced a knockout mouse inwhich the endogenous gene encoding “mouse JTT-1 antigen” wasinactivated. This knockout mouse is also useful for the above-mentionedpurpose.

[0048] The present inventions relate to polypeptides, genes, antibodies,vectors, transformants, pharmaceutical compositions, transgenic mice,knockout mice and so on, which are relevant to a novel mammalian “JTT-1antigen” isolated and identified as mentioned above. Specifically, thepresent invention are as described in (1) to (36) below.

[0049] (1) A polypeptide constituting a cell surface molecule havingcharacteristics mentioned below,

[0050] (a) said cell surface molecule is expressed in at leastthymocytes and mitogen-stimulated lymphoblast cells,

[0051] (b) an antibody reactive to said cell surface molecule inducesadhesion between mitogen-stimulated lymphoblast cells,

[0052] (c) an antibody reactive to said cell surface molecule inducesproliferation of peripheral blood lymphocytes under the coexistencewithin the presence of an antibody against CD3,

[0053] (d) said cell surface molecule has a partial amino acid sequencerepresented by Phe-Asp-Pro-Pro-Pro-Phe in its extracellular region, and

[0054] (e) said cell surface molecule has a partial amino acid sequencerepresented by Tyr-Met-Phe-Met in its cytoplasmic region.

[0055] (2) The polypeptide of (1) comprising the amino acid sequence ofSEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one ormore amino acids are substituted, deleted, or added.

[0056] (3) The polypeptide of (1), which is encoded by a DNA hybridizingwith a DNA having the nucleotide sequence of SEQ ID NO: 1 understringent conditions.

[0057] (4) The polypeptide of (1) comprising an amino acid sequencehaving 60% or more homology with an amino acid sequence of SEQ ID NO: 2.

[0058] (5) The polypeptide of any one of (1) to (4) wherein said cellsurface molecule is derived from human.

[0059] (6) A gene encoding the polypeptide of any one of (1) to (5).

[0060] (7) The gene of (6) wherein said gene is a cDNA.

[0061] (8) The gene of (7) wherein said cDNA has a nucleotide sequenceof SEQ ID NO: 1.

[0062] (9) The gene of (7) wherein said cDNA comprises a nucleotidesequence corresponding to nucleotide residues 26 to 625 of SEQ ID NO: 3,nucleotide residues 35 to 637 of SEQ ID NO: 4, nucleotide residues 1 to603 of SEQ ID NO: 5, or nucleotide residues 35 to 685 of SEQ ID NO: 6.

[0063] (10 A vector comprising the gene of any one of (6) to (9).

[0064] (11) A transformant into which the vector of (10) has beenintroduced.

[0065] (12) A transformant distinguished identified by an internationaldeposit accession No. FERM BP-5725.

[0066] (13) A polypeptide fragment comprising an extracellular region ofthe polypeptide of any one of (1) to (5).

[0067] (14) The polypeptide fragment of (13) wherein said polypeptide isa human-derived polypeptide having an amino acid sequence of SEQ ID NO:2.

[0068] (15) A gene encoding the polypeptide fragment of (13) or (14).

[0069] (16) A homodimer molecule comprising two polypeptide fragments,wherein each of the fragments comprises an extracellular region of thepolypeptide of any one of (1) to (5) and said two polypeptide fragmentsbridged through disulfide bonds to each other.

[0070] (17) The homodimer molecule of (16) wherein said polypeptide is ahuman-derived polypeptide having an amino acid sequence of SEQ ID NO: 2.

[0071] (18) A pharmaceutical composition comprising either of thepolypeptide fragment of (14) or the homodimer molecule of (17), or bothof them, and a pharmaceutically acceptable carrier.

[0072] (19) A fusion polypeptide comprising an extracellular region ofthe polypeptide of any one of (1) to (5) and a constant region of ahuman immunoglobulin (Ig) heavy chain or a portion of the constantregion.

[0073] (20) The fusion polypeptide of (19) wherein the immunoglobulin isIgG.

[0074] (21) The fusion polypeptide of (19) wherein the portion of theconstant region comprises a hinge region, C2 domain, and C3 domain ofIgG.

[0075] (22) The fusion polypeptide of any one of (19) to (21) whereinsaid polypeptide is a human-derived polypeptide having an amino acidsequence of SEQ ID NO: 2.

[0076] (23) A homodimer molecule comprising two fusion polypeptide ofany one of (19) to (22) wherein the two polypeptides bridged throughdisulfide bonds to each other.

[0077] (24) A homodimer molecule comprising two fusion polypeptides of(22) wherein the two polypeptides bridged through disulfide bonds toeach other.

[0078] (25) A pharmaceutical composition comprising either of the fusionpolypeptide of (22) or the homodimer molecule of (24), or both of them,and a pharmaceutically acceptable carrier.

[0079] (26) The pharmaceutical composition of (25) wherein saidpharmaceutical composition is utilized for treating autoimmune diseasesor allergic diseases, or for preventing said disease symptom.

[0080] (27) An antibody or a portion thereof reactive to the polypeptideof any one of (1) to (5), the polypeptide fragment of (13) or (14), orthe cell surface molecule comprising said polypeptide.

[0081] (28) The antibody of (27) or a portion of it wherein saidantibody is a monoclonal antibody.

[0082] (29) An monoclonal antibody or a portion thereof reactive to thepolypeptide having an amino acid sequence of SEQ ID NO: 2, thepolypeptide fragment of (14), or the human-derived cell surface moleculecomprising said polypeptide.

[0083] (30) A monoclonal antibody or a portion thereof reactive to thepolypeptide of any one of (1) to (5) or the cell surface moleculecomprising said polypeptide, wherein the effect of said monoclonalantibody on mitogen-stimulated lymphoblast cells is substantially thesame as the effect of a monoclonal antibody produced by a hybridomaidentified by an international deposit accession No. FERM BP-5707 onmitogen-stimulated rat lymphoblast cells.

[0084] (31) A monoclonal antibody or a portion thereof reactive to thepolypeptide of any one of (1) to (5) or the cell surface moleculecomprising said polypeptide, wherein the effect of said monoclonalantibody on mitogen-stimulated lymphoblast cells is substantially thesame as the effect of a monoclonal antibody produced by a hybridomaidentified by an international deposit accession No. FERM BP-5708 onmitogen-stimulated rat lymphoblast cells.

[0085] (32) A pharmaceutical composition comprising the monoclonalantibody of (29) or a portion thereof and a pharmaceutically acceptablecarrier.

[0086] (33) The pharmaceutical composition of (32) wherein saidpharmaceutical composition is are utilized for treating autoimmunediseases or allergic diseases, or for preventing said disease symptom.

[0087] (34) A hybridoma producing the monoclonal antibody of any one of(28) to (31).

[0088] (35) A transgenic mouse in which a gene encoding the polypeptideof (1) which is a human-derived gene comprising a nucleotide sequence ofSEQ ID NO: 1 or a rat-derived gene comprising a nucleotide sequencecorresponding to nucleotide residues 35 to 637 of SEQ ID NO: 4, which isintegrated into the mouse its endogenous gene.

[0089] (36) A knockout mouse in which its endogenous gene encoding themouse polypeptide of claim 1 comprising the amino acid sequence encodedby the gene of SEQ ID NO: 5 is inactivated so that said mousepolypeptide is not produced.

[0090] As described above, the cell surface molecule of the presentinvention (“JTT-1 antigen”) is involved in cell adhesion through themolecule, signal transmission into lymphocytes such as T cells, andfunction regulation of function of activated lymphocytes. Generalknowledge of lymphocytic cells, cell adhesion molecules, and therelationship between them and diseases are described below just forgeneral understanding of these biological events but the followinggeneral knowledge is not for interpreting the present inventionlimitedly.

[0091] Lymphocytes are roughly classified into two kinds, T cells and Bcells. After differentiation from multipotent stem cells in bone marrowto lymphoid stem cells, some of them flow into blood to reach thymus.Lymphocytes differentiated and matured in thymus, which are called Tcells (Thymus-derived T cells), get into blood again, and circulatethrough the whole body. Matured T cells have a molecule called CD3 ontheir surface. The existence of CD3 molecule is an marker to determinewhether the cells are matured T cells or not. CD3 is a convincing T cellmarker. In addition, T cells express CD4 or CD8. T cells are classifiedinto helper T cells (Th cells) assisting the antibody production by Blymphocytes, cytotoxic T cells (Tc cells, CTL) or killer T cells thatare bound to target cells to destroy them directly, suppressor T cellsthat suppress the antibody production by B lymphocytes, and effector Tcells that secrete effector substances such as lymphokines to causedelayed allergy.

[0092] B cells are derived from the lymphoid stem cells differentiatedand matured in bone marrow. B cells are those antibody-producingprecursor cells since they produce antibodies with an appropriatestimulus. B cells have immunoglobulins on their cell surface, which wereproduced in a cell. Such immunoglobulins function as receptors forantigens. Matured B cells have both IgM and IgD on their surface. If Bcells are differentiated with antigen stimulation and signals from Tcells, the production of IgM increases and their C-terminal cellmembrane binding regions are changed to be secreted. With sufficientstimulation, not only the surface immunoglobulins change into IgG, IgE,and IgA, but also the immunoglobulins of each class are secreted. Theimmunoglobulin on the B cell surface is sometimes represented as Ig,abbreviation of surface Ig, or mIg, abbreviation of membrane Ig. All Igson the surface of the same B cell have the same antigen binding sites.

[0093] There are lymphocytes called large granular lymphocytes (LGL) ornull cells, which are neither T cells nor B cells. These cells candestroy tumor cells and virus-infected cells without pre-stimulationwith antigen, which is comparative to the case of cytotoxic T cells. So,they are also called natural killer cells (NK cells).

[0094] Among the T cells mentioned above, CD4-positive T cells secretevarious cytokines, newly express receptors for these cytokines, enlargetheir own size, start cell dividing, and proliferate, when they reactwith antigen-presenting cells. Prior to these reactions at the celllevel, the complex between of the antigen peptides on antigen presentingcells and MHC class II molecules binds to the corresponding T cellantigen receptor (TCR). This causes various biochemical changes in thecells, and the signal is transmitted into nuclei to start thetranscription of specific DNAs and to produce respective proteins. As aresult, reactions at the cell level are raised. For example, cellsinfected with a virus produce virus proteins and they are degraded intopeptides by proteasomes in the cytoplasm. A part of the peptides entersendoplasmic reticulum through TAP, forms stable complex with MHC class Imolecules just produced, and transfers to the cell surface. The peptidetransferred to the cell surface is recognized specifically byCD8-positive T cells, but the T cells can not yet destroy the infectedcells at this stage. These T cells reacted to with the antigen expressesIL-2 receptor (IL-2R), are differentiated into CTL cellular cytotoxicityupon IL-2 action, and destroy their target cells to kill them in thenext time when they meet the same antigen peptide/MHC class I complex.Cytokines required for the differentiation into CTL are not only IL-2but also IFNγ or other cytokines, which are thought to have similaractions. Thus, lymphokines secreted by T cells are necessary for thedifferentiation into CTL. The lymphokines are produced as the resultthat CD4-positive Th1 cells (CD4-positive T cells secreting IL-2 orINFγ) recognize the antigen peptides derived from the same virus withclass II molecules. In some cases, without the help of CD4-positive Tcells, CD8-positive T cells react with antigens and produce IL-2 andother cytokines. When CD8-positive T cells are differentiated into CTL,granules increase in the cytoplasm. These granules comprise various highmolecular weight proteins, represented by perforin. Perforin resembles amembrane attack complex (MAC) composed of the fifth to ninth componentsof complement, and makes holes in the cell membrane of target cells. Inaddition, the granules comprise serine proteases, LT, and proteoglycan,etc. Moreover, if CD8-positive cells differentiated into CTL receiveantigen stimulation, they also secrete lymphokines such as IFNγ, LT,TNF, or IL-2. Moreover, T cells show blast transformation phenomenon,when they react with hemagglutinin (phytohemagglutinin, PHA) or ConA.

[0095] Matured T cells not yet stimulated at all are called resting Tcells, and have smaller cell size and shorter lifetime, a few days. Whenthey receive stimulation, the cells enlarge as already mentioned above,and are apt to react with various kinds of stimulation. Such T cells arecalled activated T cells. A part of the activated T cells become memoryT cells, which bring secondary immunoreaction if they receive the sameantigen stimulation. Memory T cells are thought to be kept incirculating around the body for a few years or decades.

[0096] B cells not yet stimulated at all are called resting B cells likein the case of T cells, and proliferating B cells stimulated withmultivalent antigens or CD40L, are called activated B cells. Sinceresting B cells have no costimulator molecules, which stimulate T cellswith signals through TCR, such as B7-1 (CD80) or B7-2 (CD86), presentingantigens to resting T cells are thought only to stimulate TCR and to beunable to express CD40 ligands (CD40L) or produce lymphokines.Therefore, it is thought that activated helper T cells stimulated withantigen presented by other antigen-presenting cells react with theantigen presented by resting B cells. Namely, if an antigen invades,first, dendritic cells (cells having extremely dendritic projections)expressing B7 molecules or macrophages activated by reacting withmicroorganisms present the antigen and stimulate resting helper T cellsto activate them so as to express CD40L. The activated helper T cellsthen bind to resting B cells presenting the same antigen and stimulatetheir CD40. Once B cells are activated by stimulation with multivalentantigens or CD40L, they also express B7 molecules, activate helper Tcells by stimulating CD28 on their surface with TCR, and allow thehelper T cells to express CD40L or produce lymphokines. B cells thatshow changes such as the expansion of the cell size with stimulation butnot show antibody secretion are called activated B cells. If B cells somatured meet antigens, the IgM production increases together with thestimulation from T cells and the IgM molecules so produced are secretedby turning from the membrane type into secretory type. Moreover, theyproduce isotypic antibodies other than IgM, such as IgG upon the humoralfactors from T cells. This is called isotype switching or classswitching. B cells secreting antibodies are called antibody-secretingcells. A part of them becomes morphologically characteristic cells andis called a plasma cell (Knowledge of Immunology, Ohmsha, (1996)).

[0097] Incidentally, in various reactions of immune system, thesubpopulation of white blood cells, namely, T lymphocytes, Blymphocytes, NK, neutrophils, etc., often show dynamics different fromone another. Even the same lymphocytes as mentioned above show dynamicsdifferent from one another depending on whether the cells are activatedor resting. These facts imply the existence of recognition mechanismspecific to the subpopulation of white blood cells, further, recognitionmechanism specific to the state of cells, and, in particular, celladhesion molecules (cell adhesion proteins).

[0098] Cell adhesion molecules, namely, or cell adhesion proteins are,in general, the molecules that adhere cells to each other in thedevelopment and differentiation of individuals or in migration of cellsto local site, and are known to be essential molecules for organic andfunctional contacts in a living body.

[0099] Cell adhesion molecules are roughly classified from theirstructural characteristics into five (5) families, immunoglobulinsuperfamily, integrin family, selectin family, cadherin family, and CD44family. Adhesion molecules belonging to immunoglobulin superfamily arecharacterized by the existence of repeated loop-like domains formed withdisulfide bonds. Examples thereof are intercellular adhesion molecule-1“ICAM-1” and vascular cell adhesion molecule-1 “VCAM-1.” In addition,adhesion molecules belonging to integrin family are characterized by α/βheterodimer structure. Examples thereof are “VLA-1 to 6” lymphocytefunction-associated antigen-1 “LFA-1”, “Mac-1,” and “p150/90.” Moleculesbelonging to selectin family have lectin-like domain, EGF-like domain,and complement domain in this order from N terminus. Examples thereofare “E-selectin” and “P-selectin.” Examples of cadherin family are“E-cadherin,” “N-cadherin,” and “P-cadherin,” and an example of CD44family is “CD44”.

[0100] The specific function of these adhesion molecules is known to beadhesion of white blood cells to vascular endothelial cells or oflymphocytes to antigen-presenting cells. From recent various studies, ithas been gradually revealed that adhesion molecules are involved notonly in these functions but also in various diseases.

[0101] In particular, there are many reports on diseases and expressionabnormality of adhesion molecules. For example, as for rheumatoidarthritis (RA), the expression of both “Mac-1” and “p150/95” wasreportedly strengthened in RA synoviocytes (Allen et al., ArthritisRheum., 32:947, 1989). It has also been reported that various cellsexpressed “ICAM-1” strongly and ectopically on RA synovial membrane(Hale et al., Arthritis Rheum., 32:22, 1989). Another report impliedthat “ELAM-1” was also involved in the adhesion of neutrophils tovascular endothelial cells and that the overexpression of thesemolecules was involved in infiltration of neutrophils (especially, intosynovial fluid), which is observed in RA synovial fluid (Laffon et al.,Arthritis Rheum., 32:386, 1989). Strong expression of “CD44” in vascularendothelial cells and A-type synoviocytes on RA synovial membrane wasreported (Heynes et al., Arthritis Rheum., 34:1434, 1991).

[0102] There are reports on the relationship between systemic lupuserythematosus (SLE) and the expression abnormality of adhesionmolecules. For example, adhesion ability of T lymphocytes to culturedvascular endothelial cells was reportedly lowered in SLE patients,compared to healthy volunteers. In peripheral lymphocytes of SLEpatients, adhesion molecules “ICAM-1”, “VLA-4”, and “IFA-1” to werestrongly expressed (Haskard et al., Rheumatol. Int., 9:33, 1989).

[0103] In autoimmune thyroiditis diseases, it was reported that “ICAM-1”was expressed when a thyroid follicular cells were stimulated withinterferon-γ, interleukin-1, and tumor necrosis factor, and that theformation of cluster of follicular cells and mononuclear cells wasinhibited by anti-“ICAM-1” antibody (Weetman et al., Eur. J. Immunol.,20:271, 1990).

[0104] In hepatitis, it is thought that the chances of adhesion betweenhepatocytes and inflammatory cells increases since there are twopathways of adhesion, “ICAM-1” and “LFA-3”, and “LFA-1” and “CD2”, tothereby promote presentation of antigens and activation of inflammatorycells. In particular, in hepatitis B, “LFA-3” molecules are stronglyexpressed in hepatocytes, in which viruses are actively proliferating,and “ICAM-1” well correlates with the degree of hepatitis. It is thusimplied that “LFA-3” is involved in the exclusion of viruses and“ICAM-1” promotes T cells to present antigen and regulates inflammationreaction. In “ICAM-1”-negative and HBc antigen-positive hepatocytes,chronic virus infection, a kind of immunounresponsiveness, may occur dueto no interaction between lymphocytes and hepatocytes. It has also beenreported that serum “ICAM-1” in chronic liver disease may correlate withthe degree of hepatocyte damage because the serum “ICAM-1”concentrations in acute hepatitis patients, chronic active hepatitispatients, and liver cirrhosis patients were higher than that in healthyvolunteers and chronic persisting hepatitis patients, and theconcentration was high in the case of histologically progressing activehepatitis (Mod. Phys., 15:73-76, 1995).

[0105] In a model animal of arteriosclerosis, adhesion and invasion ofmonocytes and lymphocytes to vascular endothelium were observed at veryearly stages of the onset of the disease. It is thus thought that theinteraction of these hemocytes with endothelium is the first step of theonset of arteriosclerosis. Various reports show the expression ofadhesion molecules in actual arteriosclerosis nidus including theexpression of “ICAM-1” in human arteriosclerosis nidus (Poston et al.,Am. J. Pathol., 140:665, 1992) and the expression of “VCAM-1” inarteriosclerosis nidus of a hypercholesterolemia rabbit (Cybulsky etal., Science, 251:788, 1991). A recent report revealed that theexpression of “VCAM-1” was observed in human arteriosclerosis nidus,and, in particular, strong expression in smooth muscle cells migratingto intima and in monocytes/macrophages. In addition, since theexpression of “MCP-1” was enhanced in rabbit and human arteriosclerosisnidus, suggesting that “MCP-1” promotes the formation ofarteriosclerosis nidus through the migration of monocytes/macrophages(Current Therapy 12:1485-1488, 1994).

[0106] The relationship between tumor metastasis and adhesion moleculeabnormality has also been reported. For example, if E-cadherin-decreasedcancer cells showed strong invasiveness, but the invasiveness wasinhibited by introducing the cDNA of E-cadherin into the cancer cells,the invasiveness was recovered when E-cadherin antibodies antiserum wasadded to the cells. This suggests the tight relationship between thedecrease in the expression of E-cadherin and invasiveness of tumor cells(Frixen et al., 113:173, 1991). In actual clinical cases, therelationship between the decrease of the expression of E-cadherin andmetastasis is pointed out in various kinds of cancer such as hepatoma,esophageal cancer, gastric cancer, and breast cancer. It has also beenreported that “VLA-4” molecules, a ligand for “VCAM-1”, were highlyexpressed in metastatic melanoma, gastric cancer, and breast cancer,suggesting that this molecule can could be utilized for the implantationto vascular endothelial cells in metastasis. In addition, based onexperiments using various tumor cell lines, it has been reported thatepithelial cancer, such as gastric cancer, colon large intestine cancer,lung cancer, hepatoma, or pancreatic cancer, adhered to vascularendothelial cells through E-selectin (Takada et al., Cancer Res.,53:354, 1993).

[0107] On the other hand, therapeutic approach to treat diseases bytargeting these adhesion molecules have been made. For example, it wasreported that anti-rat “ICAM-1” antibody strongly inhibited inflammatoryreaction in rat autoimmune arthritis model. It has also been reportedthat the administration of anti-“ICAM-1” antibody inhibited the onset ofarthritis in adjuvant synovitis in one of animal models of RA (Nihon etal., 14:571-577, 1991). It was further reported that the metastasisformation of inoculated tumor was remarkably inhibited if a large amountof peptides having REG sequence, which is that an amino acid sequence inan extracellular matrix protein recognized and bound by some integrins,were administered to a gallbladder cancer mouse, and that in in vitrosystem RGD peptides and anti-β1 subunit antibody inhibited the motionand infiltration of tumor cells (Yamada et al., Cancer Res., 50:4485,1990).

[0108] In the following, the present invention is described in detail byclarifying the meanings of terms used herein the present invention andthe general production methods of polypeptides, fusion polypeptides,genes, antibodies, transgenic mice, and knockout mice of the presentinvention. However, it is needless to say that the meanings of the termsare not to be interpreted limitedly by the definition given herein.

[0109] “Mitogen” used herein is also called also mitogenic factor andmeans a substance which induces cell division. Immunologically, it meansa substance inducing blastogenesis of lymphocytes polyclonally andinducing cell division. Examples thereof are lectins such as PHA andPWM, Concanavalin A (ConA), lipopolysaccharides, streptolysin S, andanti-lymphocyte antibody. It is known that Concanavalin A and PHA actonly on T lymphocytes, that lipopolysaccharides act only on Blymphocytes, and that PWM acts on both lymphocytes.

[0110] The term “lymphoblast cell” used herein is also called also alarge lymphocyte, lymphoblast, or immunoblast, and means a lymphocytebelonging to a large lymphocyte among lymphocytes existing in lymphoidtissues (lymph node, spleen, thymus, bone marrow, lymph duct, tonsil,etc.) and blood.

[0111] The term “activated lymphocyte” used herein, for example, alymphocyte mentioned below, but is not limited thereto. For example, theterm means a lymphocyte activated by some stimulation. As mentionedabove, lymphocytes are classified into T cells, B cells, and naturalkiller cells. T cells are classified into CD4-positive cells andCD8-positive cells. Therefore, the “activated lymphocytes” of thepresent invention include mainly activated T cells, activated B cells,and activated natural killer cells, and activated T cells includeactivated CD4-positive cells and activated CD8-positive cells.

[0112] Upon reacting with antigens presented by antigen-presentingcells, CD4-positive T cells secrete various cytokines, newly expressreceptors for these cytokines, enlarge their own size, start celldividing, proliferate, and are activated. Activated CD4-positive T cellsinclude those in such a state.

[0113] CD8-positive T cells express IL-2R when they react with antigens.When IL-2 acts on IL-2R, the cells are differentiated into CTL, whichhas cellular cytotoxicity. CTL destroy their its target cells to killthem when they meet the same antigen peptide/MHC class I complex. WhenCD8-positive T cells are differentiated into CTL, granules increase inthe cytoplasm. These granules comprise various high molecular weightproteins, represented by perforin. Perforin resembles MAC composed ofthe fifth to ninth components of complement, and makes holes in the cellmembrane of target cells. The granules also comprise serine proteases,LT, and proteoglycan. If CD8-positive cells receive antigen stimulationand are differentiated into CTL, they also secrete lymphokines such asIFNγ, LT, TNF, or IL-2. Activated CD8-positive T cells include those insuch a state.

[0114] T cells show blast formation phenomenon when they react withhemagglutinin (phytohemagglutinin, PHA) or Concanavalin A (ConA).Activated T cells comprise include those in such a state.

[0115] B cells express B7 molecules, activate helper T cells bystimulating CD28 on their surface with TCR, allow the helper T cells toexpress CD40L or produce lymphokines. When the cells receivestimulation, they change to expand their cell size or proliferate.Activated B cells include those in such a state. In the presentinvention, activated B cells include those secreting antibodies(antibody-secreting cells and plasma cells).

[0116] Activated natural killer cells mean those showing cytotoxicaction on tumor cells or virus-infected cells as mentioned above. In thepresent invention, activated lymphocytes include thymus cells stimulatedby Concanavalin A (ConA).

[0117] The “activated lymphoblast cell” used herein includes anactivated “lymphoblast” that is generated when the lymphoblast mentionedabove is stimulated with “mitogen” mentioned above such as ConcanavalinA.

[0118] The term “resting lymphocyte” used herein, in some case, annon-activated lymphocyte, which has not received the stimulation toactivate cells, in contrast to an activated lymphocyte mentioned above.

[0119] The “gene” of the present invention includes a genomic DNA and acDNA.

[0120] The “human-derived” substance of the present invention includesnatural substance isolated from a human body component (organ, tissue,cell, body fluid, etc.), and recombinant substance produced byrecombinant DNA technology. When the substance is protein orpolypeptide, the substance includes an artificial protein andpolypeptide having an amino acid sequence where one or more amino acidsare substituted, deleted, or added.

[0121] The “cell surface molecule” of the present invention is thatderived from a mammal such as human, rat, mouse, guinea pig, and rabbit,preferably that derived from human, rat, or mouse, and more preferablythat derived from human.

[0122] Specifically, the “cell surface molecule” of the presentinvention is that characterized by having, at least, propertiesdescribed below.,

[0123] (a) the cell surface molecule is expressed in, at least,thymocytes and mitogen-stimulated lymphoblast cells;

[0124] (b) an antibody reactive to the cell surface molecule inducesadhesion between mitogen-stimulated lymphoblast cells;

[0125] (c) an antibody reactive to the cell surface molecule inducesproliferation of peripheral blood lymphocytes under the coexistencewithin the presence of an antibody against CD3;

[0126] (d) the cell surface molecule has a partial amino acid sequencerepresented by Phe-Asp-Pro-Pro-Pro-Phe in its extracellular region; and

[0127] (e) the cell surface molecule has a partial amino acid sequencerepresented by Tyr-Met-Phe-Met in its cytoplasmic region.

[0128] Preferably, the “cell surface molecule” comprises the following“polypeptide” of the present invention.

[0129] The “polypeptide” of the present invention is that whichconstitutes the above-mentioned “cell surface molecule” of the presentinvention. Examples thereof are as follows.

[0130] (1) A polypeptide encoded by a DNA hybridizing with a DNAcomprising a nucleotide sequence of SEQ ID NO: 1 under stringentconditions;

[0131] (2) A polypeptide having an amino acid sequence having 60% ormore homology with an amino acid sequence of SEQ ID NO: 2;

[0132] (3) A polypeptide having an amino acid sequence of SEQ ID NO: 2or an amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting “human JTT-1 antigen” andits derivative);

[0133] (4) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 26 to 625 ofSEQ ID NO: 3 or an amino acid sequence substantially the same as theamino acid sequence (namely, a polypeptide constituting “human JTT-1antigen” and its derivative);

[0134] (5) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 35 to 637 ofSEQ ID NO: 4 or an amino acid sequence substantially the same as theamino acid sequence (namely, a polypeptide constituting “rat JTT-1antigen” and its derivative);

[0135] (6) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 1 to 603 of SEQID NO: 5 or an amino acid sequence substantially the same as the aminoacid sequence (namely, a polypeptide constituting “mouse JTT-1 antigen”and its derivative);

[0136] (7) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 35 to 685 ofSEQ ID NO: 6 or an amino acid sequence substantially the same as theamino acid sequence (namely, a polypeptide constituting a “mutant of ratJTT-1 antigen” and its derivative); and

[0137] (8) A polypeptide having an amino acid sequence encoded by a DNAencoding a polypeptide constituting the cell surface molecule of thepresent invention, wherein the DNA is introduced into the transformantidentified by an international deposit accession No. FERM BP-5725 or,having amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting a “human JTT-1 antigen” andits derivative).

[0138] To determine the “percent homology” of two amino acid sequencesor of two nucleic acids, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent homology between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions (e.g., overlapping positions)×100). In one embodiment the twosequences are the same length.

[0139] To determine percent homology between two sequences, thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877 is used. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.Biol. 215:403-410. BLAST nucleotide searches are performed with theNBLAST program, score=100, word length=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches are performed with the XBLAST program, score=50, word length=3to obtain amino acid sequences homologous to a VRK1 or VRK2 proteinmolecules. To obtain gapped alignments for comparison purposes, GappedBLAST is utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used. See http://www.ncbi.nlm.nih.gov.

[0140] Furthermore, the present invention relates to a DNA thatspecifically hybridizes under moderate or highly stringent conditions toa DNA encoding a protein of the present invention and comprises at least15 nucleotide residues. The DNA can be used, for example, as a probe todetect or isolate a DNA encoding a protein of the present invention, oras a primer for PCR amplification. An example is DNA consisting of atleast 15 nucleotides complementary to the nucleotide sequence of SEQ IDNO: 1, NO: 3, NO:4, NO:5 or NO:6.

[0141] Standard hybridization conditions (e.g., moderate or highlystringent conditions) are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6, hereby incorporated by reference. Moderatehybridization conditions are defined as equivalent to hybridization in2× sodium chloride/sodium citrate (SSC) at 30° C., followed by one ormore washes in 1×SSC, 0.1% SDS at 50-60° C. Highly stringent conditionsare defined as equivalent to hybridization in 6× sodium chloride/sodiumcitrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 50-65° C.

[0142] Examples of “stringent conditions” are as follows. When a probewith 50 or more nucleotides is used and hybridization is performed in0.9% NaCl, the standard of temperature where 50% dissociation occurs(Tm) is calculated using the following formula and the temperature forhybridization can be determined according to the following formula.

Tm=82.3° C.+0.41×(G+C) %−500/n−0.61×(formamide) %

[0143] (n means the number of the nucleotide of probe).

[0144] Temperature=Tm−25° C.

[0145] In addition, when a probe with 100 or more nucleotides (G+C=40 to−50%) is used, it should be considered that Tm varies as (1) and (2)mentioned below.

[0146] (1) Tm descends by about 1° C. per 1% mismatch.

[0147] (2) Tm descends by 0.6 to 0.7° C. per 1% formamide.

[0148] Accordingly, the temperature conditions for the combination ofcompletely complementary strands can be set as follows.

[0149] (A) 65 to 75° C. (formamide not added)

[0150] (B) 35 to 45° C. (in the presence of 50% formamide)

[0151] The temperature conditions for the combination of incompletelycomplementary strands can be set as follows.

[0152] (A) 45 to 55° C. (formamide not added

[0153] (B) 35 to 42° C. (in the presence of 30% formamide)

[0154] The temperature conditions when a probe with 23 or lessnucleotides is used can be 37° C. or can be calculated using thefollowing formula.

Temperature=2° C.×(the number of A+T)+4° C.×(the number of C+G)−5° C.

[0155] Here, “having substantially the same amino acid sequence” meansto include a polypeptide having an amino acid sequence where multipleamino acids, preferably 1 to 10 amino acids, particularly preferably 1to 5 amino acids, in the amino acid sequence shown in Sequence Listingare substituted, deleted, and/or modified, and a polypeptide having anamino acid sequence where multiple amino acids, preferably 1 to 10 aminoacids, particularly preferably 1 to 5 amino acids, are added to theamino acid sequence shown in Sequence Listing, as long as thepolypeptide has substantially the same biological properties as thepolypeptide having the amino acid sequence shown in Sequence Listing.

[0156] Such substitution, deletion, or insertion of amino acids can beperformed by the usual method (Experimental Medicine: SUPPLEMENT,“Handbook of Genetic Engineering” (1992); and so on).

[0157] Examples thereof are synthetic oligonucleotide-directedmutagenesis (gapped duplex method), point metagenesis by which a pointmutation is introduced at random by treatment with nitrite or sulfite,the method by which a deletion mutant is prepared with Bal31 enzyme andthe like, cassette mutagenesis, linker scanning method, missincorporation method, mismatch primer method, DNA segment synthesismethod, etc.

[0158] Synthetic oligonucleotide-directed mutagenesis (gapped duplexmethod) can be, for example, performed as follows. The region desired tobe mutagenized is cloned into M13 phage vector having amber mutation toprepare the single-stranded phage DNA. After RF I DNA of M13 vectorwithout amber mutation is linearized by restriction enzyme treatment,DNA is mixed with the single-stranded phage DNA mentioned above,denatured, and annealed thereby forming “gapped duplex DNA.” A syntheticoligonucleotide into which mutations are introduced is hybridized withthe gapped duplex DNA and the closed-circular double-stranded DNAs areprepared by the reactions with DNA polymerase and DNA ligase. E. colimutS cells, deficient in mismatch repair activity, are transfected withthis DNA., E. coli cells without suppressor activity are infected withthe grown phages, and only phages without amber mutation are screened.

[0159] The method by which a point mutation is introduced with nitriteutilizes, for example, the principle as mentioned below. If DNA istreated with nitrite, bases are deaminated to change adenine intohypoxanthine, cytosine into uracil, and guanine into xanthine. Ifdeaminated DNA is introduced into cells, “A:T” and “G:C” are replacedwith “G:C” and “A:T”, respectively, because hypoxanthine, uracil, andxanthine form a base pair with cytosine, adenine, and thymine,respectively, in the DNA replication. Actually, single-stranded DNAfragments treated with nitrite are hybridized with “gapped duplex DNA”,and thereafter mutant strains are separated by manipulating in the sameway as synthetic oligonucleotide-directed mutagenesis (gapped duplexmethod).

[0160] Conservative amino acid substitutions can also be made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

[0161] Alphabetical triplet or single letter codes used to representamino acids in the present specification or figures mean amino acids asfollows. (Gly/G) glycine, (Ala/A) alanine, (Val/V) valine, (Leu/L)leucine, (Ile/I) isoleucine, (Ser/S) serine, (Thr/T) threonine, (Asp/D)aspartic acid, (Glu/E) glutamic acid, (Asn/N) asparagine, (Gln/Q)glutamine, (Lys/K) lysine, (Arg/R) arginine, (Cys/C) cysteine, (Met/M)methionine, (Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W)tryptophane, (His/H) histidine, (Pro/P) proline.

[0162] The “polypeptide” constituting the above-mentioned “cell surfacemolecule” of the present invention is a transmembrane protein, whichpenetrates cell membrane, and the “cell surface molecule” is composed ofone or two of these transmembrane polypeptides.

[0163] Here, a “transmembrane protein” means a protein that connectswith membrane through the hydrophobic peptide region penetrating thelipid bilayer of the membrane once or several times and whose structureis, as a whole, composed of three main regions, that is, extracellularregion, transmembrane region, and cytoplasmic region, as seen in manyreceptors or cell surface molecules. Such a transmembrane proteinconstitutes each receptor or cell surface molecule by existing in theform of a monomer, homodimer, heterodimer or oligomer with anotherchain(s) having the same or different amino acid sequence.

[0164] The “polypeptide fragment” of the present invention is a fragmentfrom the above-defined “polypeptide” of the present invention, andpreferably the extracellular region of the polypeptide. One to fiveamino acids, if desired, can be added to the N terminus and/or Cterminus of this region.

[0165] Here, an “extracellular region” means the whole or a portion fromthe partial structure (partial region) from the entire structure of theabove-mentioned transmembrane protein where the partial structure existsoutside of the membrane. In other words, it means the whole or a portionof the region of the transmembrane protein except the region integratedincorporated into the membrane (transmembrane region) and the regionexisting in the cytoplasm following the transmembrane region in themembrane (cytoplasmic regions).

[0166] “The constant region or a portion of the constant region of humanimmunoglobulin (Ig) heavy chain” used herein means the constant regionor the Fc region of human-derived immunoglobulin heavy chain (H chain)as described above, or a portion of them. The immunoglobulin can be anyimmunoglobulin belonging to any class and any subclass. Specifically,examples of the immunoglobulin are IgG (IgG1, IgG2, IgG3, and IgG4),IgM, IgA (IgA1 and IgA2), IgD, and IgE. Preferably, the immunoglobulinis IgG (IgG1, IgG2, IgG3, or IgG4), or IgM. Examples of particularlypreferable immunoglobulin in of the present invention are thosebelonging to human-derived IgG (IgG1, IgG2, IgG3, or IgG4).

[0167] Immunoglobulin has a Y-shaped structural unit in which fourchains composed of two homologous light chains (L chains) and twohomologous heavy chains (H chains) are connected through disulfide bonds(S—S bonds). The light chain is composed of the light chain variableregions (VL) and the light chain constant region (CL). The heavy chainis composed of the heavy chain variable regions (VH) and the heavy chainconstant region (CH).

[0168] The heavy chain constant region is composed of some domainshaving the amino acid sequences inherent in each class (IgG, IgM, IgA,IgD, and IgE) and each subclass (IgG1, IgG2, IgG3, and IgG4, IgA1, andIgA2).

[0169] The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is composedof VH, CH1 domain, hinge region, CH2 domain, and CH3 domain in thisorder from N terminus.

[0170] Similarly, the heavy chain of IgG1 is composed of VH, Cγ₁1domain, hinge region, Cγ₁2 domain, and Cγ₁3 domain in this order from Nterminus. The heavy chain of IgG2 is composed of VH, Cγ₂2 domain, hingeregion, Cγ₂2 domain, and Cγ₂3 domain in this order from N terminus. Theheavy chain of IgG3 is composed of VH, Cγ₃1 domain, hinge region, Cγ₃2domain, and Cγ₃3 domain in this order from N terminus. The heavy chainof IgG4 is composed of VH, Cγ₄1 domain, hinge region, Cγ₄2 domain, andCγ₄3 domain in this order from N terminus.

[0171] The heavy chain of IgA is composed of VH, Cα1 domain, hingeregion, Cγ₁2 domain, and Cγ₃3 domain in this order from N terminus.

[0172] Similarly, the heavy chain of IgA1 is composed of VH, Cα₁1domain, hinge region, Cα₁2 domain, and Cα₁3 domain in this order from Nterminus. The heavy chain of IgA2 is composed of VH, Cα₂1 domain, hingeregion, Cα₂2 domain, and Cα₂3 domain in this order from N terminus.

[0173] The heavy chain of IgD is composed of VH, Cδ1 domain, hingeregion, Cδ2 domain, and Cδ3 domain in this order from N terminus.

[0174] The heavy chain of IgM is composed of VH, Cε1 domain, Cε2 domain,Cε3 domain, and Cε4 domain in this order from N terminus and have nohinge region as seen in IgG, IgA, and IgD.

[0175] The heavy chain of IgE is composed of VH, Cε1 domain, CEε2domain, Cε3 domain, and Cε4 domain in this order from N terminus andhave no hinge region as seen in IgG, IgA, and IgD.

[0176] If, for example, IgG is treated with papain, it is cleaved at theslightly N terminal side beyond the disulfide bonds existing in thehinge region where the disulfide bonds connect the two heavy chains togenerate two homologous Fab, in which a heavy chain fragment composed ofVH and CH1 is connected with one light chain through a disulfide bond,and one Fc, in which two homologous heavy chain fragments composed ofthe hinge region, CH2 domain, and CH3 domain are connected throughdisulfide bonds (See “Immunology Illustrated”, original 2nd ed.,Nankodo, pp. 65-75 (1992); and “Focus of Newest Medical Science‘Recognition Mechanism of Immune System’”, Nankodo, pp. 4-7 (1991); andso on).

[0177] Namely, “a portion of a constant region of immunoglobulin heavychain” of the present invention means a portion of a constant region ofan immunoglobulin heavy chain having the structural characteristics asmentioned above, and preferably, is the constant region without C1domain, or the Fc region. Specifically, examples thereof are the regioncomposed of hinge region, C2 domain, and C3 domain in the case from eachof IgG, IgA, and IgD, and are the region composed of C2 domain, C3domain, and C4 domain in the case from each of IgM and IgE. Aparticularly preferable example thereof is the Fc region ofhuman-derived IgG1.

[0178] The “fusion polypeptide” of the present invention is thatcomposed of the extracellular region of the “polypeptide” constitutingthe above-described “cell surface molecule” of the present invention and“a constant region or a portion of a constant region of humanimmunoglobulin (Ig) heavy chain.” Preferably, it is a fusion polypeptidecomposed of an extracellular region of a polypeptide of the presentinvention and a portion of a constant region of human IgG heavy chain,and particularly preferably, it is a fusion polypeptide composed of anextracellular region of a polypeptide of the present invention and theregion (Fc) composed of a hinge region, CH2 domain, and CH3 domain ofhuman IgG heavy chain. Moreover, IgG1 is preferable among IgG. Inaddition, a polypeptide derived from human, mouse, or rat (preferably,human) is preferable as the polypeptide of the present invention.

[0179] The fusion polypeptide of the present invention has the advantagethat the fusion polypeptide can be purified extremely easily by usingaffinity column chromatography using the property of protein A, whichbinds specifically to the immunoglobulin fragment because the fusionpolypeptide of the present invention has a portion of a constant region(for example Fc) of an immunoglobulin such as IgG as mentioned above asa fusion partner. Moreover, since various antibodies against the Fc ofvarious immunoglobulin are available, an immunoassay for the fusionpolypeptides can be easily performed with antibodies against the Fc.

[0180] The polypeptide, polypeptide fragment, and fusion polypeptide ofthe present invention can be produced not only by recombinant DNAtechnology as mentioned below but also by a method well known in the artsuch as a chemical synthetic method and a cell culture method, or amodified method thereof.

[0181] The “gene” of the present invention comprises a DNA encoding theabove-mentioned polypeptide or polypeptide fragment of the presentinvention, and includes any gene having a nucleotide sequence encodingthe polypeptide or polypeptide fragment of the present invention.

[0182] Examples of the gene are those encoding the polypeptide orpolypeptide fragment mentioned below.

[0183] (1) A polypeptide encoded by a DNA hybridizing with a DNAcomprising a nucleotide sequence of SEQ ID NO: 1 under stringentconditions;

[0184] (2) A polypeptide having an amino acid sequence having 60% ormore homology with an amino acid sequence of SEQ ID NO: 2;

[0185] (3) A polypeptide having an amino acid sequence of SEQ ID NO: 2or an amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting “human JTT-1 antigen” andits derivative);

[0186] (4) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 26-625 of SEQID NO: 3 or an amino acid sequence substantially the same as the aminoacid sequence (namely, a polypeptide constituting “human JTT-1 antigen”and its derivative);

[0187] (5) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 35-637 of SEQID NO: 4 or an amino acid sequence substantially the same as the aminoacid sequence (namely, a polypeptide constituting “rat JTT-1 antigen”and its derivative);

[0188] (6) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 1-603 of SEQ IDNO: 5 or an amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting “mouse JTT-1 antigen” andits derivative);

[0189] (7) A polypeptide having an amino acid sequence encoded by anucleotide sequence corresponding to nucleotide residues 35-685 of SEQID NO: 6 or an amino acid sequence substantially the same as the aminoacid sequence (namely, a polypeptide constituting a “mutant of rat JTT-1antigen” and its derivative); and

[0190] (8) A polypeptide having an amino acid sequence encoded by a DNAencoding a polypeptide constituting the cell surface molecule of thepresent invention, wherein the DNA is introduced into the transformantidentified by an international deposit accession No. FERM BP-5725 or,having an amino acid sequence substantially the same as said amino acidsequence (namely, a polypeptide constituting a “human JTT-1 antigen” andits derivative).

[0191] Here, “substantially the same amino acid sequence” means asdefined above.

[0192] Specific examples of the gene of the present invention are DNAsor their fragments mentioned below.

[0193] (1) A DNA comprising a nucleotide sequence of SEQ ID NO: 1, and aDNA hybridizing with the DNA under stringent conditions;

[0194] (4) A DNA comprising a nucleotide sequence corresponding tonucleotide residues 26-625 of SEQ ID NO: 3;

[0195] (5) A DNA comprising a nucleotide sequence corresponding tonucleotide residues 35-637 of SEQ ID NO: 4;

[0196] (6) A DNA comprising a nucleotide sequence corresponding tonucleotide residues 1-603 of SEQ ID NO: 5;

[0197] (7) A DNA comprising a nucleotide sequence corresponding tonucleotide residues 35-685 of SEQ ID NO: 6;

[0198] (8) A DNA encoding a polypeptide constituting a cell surfacemolecule of the present invention, wherein the DNA is introduced into atransformant identified by an international deposit accession No. FERMBP-5725.

[0199] The DNA encoding a portion of a constant region of immunoglobulinheavy chain, which is a part of a fusion polypeptide of the presentinvention, can be cDNA, or genomic DNA comprised of intons between everyexon (the DNA encoding, for example, CH1 domain, hinge region, CH2domain, CH3 domain, CH4 domain and so on).

[0200] The DNA of the present invention includes any DNA comprised ofany codons as long as the codons encode the same amino acids.

[0201] The DNA of the present invention can be a DNA obtained by anymethod. For example, the DNA includes complementary DNA (cDNA) preparedfrom mRNA, DNA prepared from genomic DNA, DNA prepared by chemicalsynthesis, DNA obtained by PCR amplification with RNA or DNA as atemplate, and DNA constructed by appropriately combining these methods.

[0202] The DNA encoding the polypeptide of the present invention can beobtained by the usual method such as a method to clone cDNA from mRNAencoding the polypeptide of the present invention, a method to isolategenomic DNA and then splice them, chemical synthesis and so on.

[0203] (1) cDNA can be cloned from the mRNA encoding the polypeptide ofthe present invention by, for example, the method described below.

[0204] First, the mRNA encoding a cell surface molecule (polypeptide) ofthe present invention is prepared from tissues or cells (for example,thymus cells or spleen-derived lymphoblast cells stimulated with ConA)expressing and producing a cell surface molecule (polypeptide) of thepresent invention. mRNA can be prepared isolating total RNA by a knownmethod such as quanidine-thiocyanate method (Chirgwin et al.,Biochemistry, 18:5294, 1979), hot phenol method, or AGPC method, andsubjecting it to affinity chromatography using oligo-dT cellulose orpoly-U Sepharose.

[0205] Then, with the mRNA obtained as a template, cDNA is synthesized,for example, by a well-known method using reverse transcriptase such asthe method of Okayama et al. (Mol. Cell. Biol. 2:161, 1982; ibid. 3:280,1983) or the method of Hoffman et al. (Gene 25:263, 1983), and convertedinto double-stranded cDNA. A cDNA library is prepared by transforming E.coli with plasmid vectors, phage vectors, or cosmid vectors having thiscDNA or by transfecting E. coli after in vitro packaging.

[0206] The plasmid vectors used in this invention are not limited aslong as they are replicated and maintained in hosts. Any phage vectorsthat can be replicated in hosts can also be used. Examples of usuallyused cloning vectors are pME18S, λZAPII (1ZAPII), pUC19, λgt10, λgt11,and so on. When the vector is applied to immunological screening asmentioned below, the vector having a promoter that can express a geneencoding the polypeptide of the present invention in a host ispreferably used.

[0207] cDNA can be inserted into a plasmid by, for example, the methodof Maniatis et al. (Molecular Cloning, A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory, p. 1.53, 1989). cDNA can beinserted into a phage vector by, for example, the method of Hyunh et al.(DNA cloning, a practical approach, Vol. 1, p. 49 (1985)). These methodscan be simply performed by using a commercially available cloning kit(for example, a product from Takara Shuzo). The recombinant plasmid orphage vector thus obtained is introduced into appropriate host cellssuch as a prokaryote (for example, E. coli: XL1Blue MRF′, DH5α, HB101,MC1061/P3, etc.).

[0208] Examples of a method for introducing a plasmid into a host arecalcium chloride method, calcium chloride/rubidium chloride methoddescribed in Molecular Cloning, A Laboratory Manual (second edition,Cold Spring Harbor Laboratory, p. 1.74 (1989)), and electroporationmethod. Phage vectors can be introduced into host cells by, for example,a method in which the phage DNAs are introduced into grown hosts afterin vitro packaging. In vitro packaging can be easily performed with acommercially available in vitro packaging kit (for example, a productfrom Stratagene or Amersham).

[0209] The cDNA encoding the polypeptide of the present invention can beisolated from the cDNA library so prepared according to the methodmentioned above by combining general cDNA screening methods.

[0210] For example, a clone comprising the desired cDNA can be screenedby a known colony hybridization method (Crunstein et al., Proc. Natl.Acad. Sci. USA, 72:3961, 1975) or plaque hybridization method (MolecularCloning, A Laboratory Manual, second edition, Cold Spring HarborLaboratory, p. 2.108 (1989)) using ³²P-labeled chemically synthesizedoligonucleotides as probes, which are corresponding to the amino acidsequence of the polypeptide of the present invention. Alternatively, aclone having a DNA fragment encoding a specific region within thepolypeptide of the present invention can be screened by amplifying theregion by PCR with synthetic PCR primers.

[0211] When a cDNA library prepared using a cDNA expression vector (forexample, λZAPII phage vector) is used, the desired clone can be screenedby the antigen-antibody reaction using an antibody against thepolypeptide of the present invention. A screening method using PCRmethod is preferably used when many clones are subjected to screening.

[0212] The nucleotide sequence of the DNA thus obtained can bedetermined by Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci.USA, 74:560, 1977) or the dideoxynucleotide synthetic chain terminationmethod using phage M13 (Sanger et al., Proc. Natl. Acad. Sci. USA,74:5463-5467, 1977). The whole or a portion of the gene encoding thepolypeptide of the present invention can be obtained by excising theclone obtained as mentioned above with restriction enzymes and so on.

[0213] (2) The DNA encoding the polypeptide of the present invention canbe isolated from the genomic DNA derived from the cells expressing thepolypeptide of the present invention as mentioned above by the followingmethods.

[0214] Such cells are solubilized preferably by SDS or proteinase K, andthe DNAs are deproteinized by repeating phenol extraction. RNAs aredigested preferably with ribonuclease. The DNAs obtained are partiallydigested with appropriate restriction enzymes, and the DNA fragmentsobtained are amplified with appropriate phage or cosmid to generate alibrary. Then, clones having the desired sequence are detected, forexample, by using radioactively labeled DNA probes, and the whole or aportion of the gene encoding the polypeptide of the present invention isobtained from the clones by excision with restriction enzyme and so on.

[0215] cDNA encoding a human-derived polypeptide can be obtained asfollows. After a cosmid library into which human genomic DNA(chromosomal DNA) is introduced is prepared (“Laboratory Manual: HumanGenome Mapping”, Maruzen press), positive clones comprising the DNA ofthe coding region of the desired protein are obtained by screening thecosmid library. Then, the cDNA library mentioned above is screened withthe coding DNA excised from the positive clone as a probe to prepare thehuman cDNA.

[0216] (3) The DNA of the present invention can also be chemicallysynthesized by the usual method, based on the nucleotide sequence of SEQID NO: 1, 3, 4, 5, or 6.

[0217] The present invention also relates to a recombinant vectorcomprising the DNA encoding an above-mentioned cell surface molecule(polypeptide) of the present invention. The recombinant vector of thepresent invention is not limited as long as it can be replicated andmaintained or can autonomously replicate in various prokaryotic and/oreukaryotic hosts. The vector of the present invention includes plasmidvectors and phage vectors.

[0218] The recombinant vector can easily be prepared by ligating the DNAencoding the polypeptide of the present invention with a vector forrecombination available in the art (plasmid DNA and bacteriophage DNA)by the usual method. Specific examples of the vectors for recombinationused are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13,and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillussubtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples ofphages are a bacteriophage such as λ phage, and an animal or insectvirus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, andnuclear polyhidrosis virus.

[0219] An expression vector is useful for expressing the DNA encodingthe polypeptide of the present invention and for producing thepolypeptide of the present invention. The expression vector is notlimited as long as it expresses the gene encoding the polypeptide of thepresent invention in various prokaryotic and/or eukaryotic host cellsand produces this protein. Examples thereof are pEFneo (Proc. Natl.Acad. Sci. USA 91:158-162, 1994), PEF-BOS (Nucleic Acids Res. 18:5322,1990), pME18S (Experimental Medicine: SUPPLEMENT, “Handbook of GeneticEngineering” (1992)), pMAL C2, and so on.

[0220] When bacteria, particularly E. coli are used as host cells, anexpression vector is generally comprised of, at least, apromoter/operator region, an initiation codon, the DNA encoding thepolypeptide of the present invention, termination codon, terminatorregion, and replicon.

[0221] When yeast, animal cells, or insect cells are used as hosts, anexpression vector is preferably comprised of, at least, a promoter, aninitiation codon, the DNA encoding the polypeptide of the presentinvention, and a termination codon. It may also comprise the DNAencoding a signal peptide, enhancer sequence, 5′- and 3′-untranslatedregion of the gene encoding the polypeptide of the present invention,splicing junctions, polyadenylation site, selectable marker region, andreplicon. The expression vector may also contain, if required, a genefor gene amplification (marker) that is usually used.

[0222] A promoter/operator region to express the polypeptide of thepresent invention in bacteria comprises a promoter, an operator, and aShine-Dalgarno (SD) sequence (for example, AAGG). For example, when thehost is Escherichia, it preferably comprises Trp promoter, lac promoter,recA promoter, λPL promoter, lpp promoter, tac promoter, or the like.Examples of a promoter to express the polypeptide of the presentinvention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADHpromoter, and so on. When the host is Bacillus, examples thereof areSL01 promoter, SP02 promoter, penP promoter and so on. When the host isa eukaryotic cell such as a mammalian cell, examples thereof areSV40-derived promoter, retrovirus promoter, heat shock promoter, EFpromoter, and so on, and preferably SV40, SRα, and retrovirus-derivedone. As a matter of course, the promoter is not limited to the aboveexamples. In addition, to use an enhancer is effective for expression.

[0223] A preferable initiation codon is, for example, a methionine codon(ATG).

[0224] The commonly used termination codon (for example, TAG, TGA, TAA,and so on) is illustrated as a termination codon.

[0225] Usually used natural or synthetic terminators are used as aterminator region.

[0226] A replicon means a DNA capable of replicating the whole DNAsequence in host cells, and includes a natural plasmid, an artificiallymodified plasmid (DNA fragment prepared from a natural plasmid), asynthetic plasmid, and so on. Examples of a preferable plasmids arepBR322 or its artificial derivatives (DNA fragment obtained by treatingpBR322 with appropriate restriction enzymes) for E. coli, yeast 2μplasmid or yeast chromosomal DNA for yeast, and pEFneo, pME18S, pRSVneoATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC37149, etc., for mammalian cells.

[0227] An enhancer sequence, polyadenylation site, and splicing junctionthat are usually used in the art, such as those derived from SV40 can bealso used.

[0228] A selectable marker usually used can be used according to theusual method. Examples thereof are resistance genes for antibiotics,such as tetracycline, neomycin, ampicillin, or kanamycin, and thymidinekinase gene.

[0229] Examples of a gene for gene amplification are dihydrofolatereductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene,glutamate synthase gene, adenosine deaminase gene, ornithinedecarboxylase gene, hygromycin-B-phophotransferase gene, aspartatetranscarbamylase gene, etc.

[0230] The expression vector of the present invention can be prepared bycontinuously and circularly linking at least the above-mentionedpromoter, initiation codon, DNA (gene) encoding the polypeptide of thepresent invention, termination codon, and terminator region, to anappropriate replicon. If desired, appropriate DNA fragments (forexample, linkers, restriction sites generated with other restrictionenzyme), can be used by the usual method such as digestion with arestriction enzyme or ligation using T4 DNA ligase.

[0231] Transformants of the present invention can be prepared byintroducing the expression vector mentioned above into host cells.

[0232] Host cells used in the present invention are not limited as longas they are compatible with an expression vector mentioned above and canbe transformed. Examples thereof are various cells such as natural cellsor artificially established recombinant cells usually used in technicalfield of the present invention (for example, bacteria (Escherichia andBacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, or insectcells.

[0233]E. coli or animal cells are preferably used. Specific examples areE. coli (DH5α, XL1Blue MRF′, TB1, HB101, etc.), mouse-derived cells(COP, L, C127, Sp2/0, NS1, NIH 3T3, etc.), rat-derived cells,hamster-derived cells (BHK, CHO-K1, CHO, etc.), monkey-derived cells(COS1, COS3, COS7, CV1, Velo, etc.), and human-derived cells (HEK293,Hela, diploid fibroblast-derived cells, myeloma, Namalwa, etc.).

[0234] An expression vector can be introduced (transformed (transduced))into host cells by known method.

[0235] Transformation can be performed, for example, according to themethod of Cohen et al. (Proc. Natl. Acad. Sci. USA 69:2110, 1972),protoplast method (Mol. Gen. Genet. 168:111, 1979), or competent method(J. Mol. Biol. 56:209, 1971) when the hosts are bacteria (E. coli,Bacillus subtilis, etc.), the method of Hinnen et al. (Proc. Natl. Acad.Sci. USA 75:1927, 1978), or lithium method (J. Bacteriol. 153:163, 1983)when the host is Saccharomyces cerevisiae, the method of Graham(Virology 52:456, 1973) when the hosts are animal cells, and the methodof Summers et al. (Mol. Cell. Biol. 3:2156-2165, 1983) when the hostsare insect cells.

[0236] The polypeptide of the present invention can be produced bycultivating transformants (in the following this term includestransductants) comprising an expression vector prepared as mentionedabove in nutrient media.

[0237] The nutrient media preferably comprise carbon source, inorganicnitrogen source, or organic nitrogen source necessary for the growth ofhost cells (transformants). Examples of the carbon source are glucose,dextran, soluble starch, and sucrose, and examples of the inorganic ororganic nitrogen source are ammonium salts, nitrates, amino acids, cornsteep liquor, peptone, casein, meet extract, soy bean cake, and potatoextract. If desired, they may comprise other nutrients (for example, aninorganic salt (for example, calcium chloride, sodiumdihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (forexample, tetracycline, neomycin, ampicillin, kanamycin, etc.).

[0238] Cultivation is performed by a method known in the art.Cultivation conditions such as temperature, pH of the media, andcultivation time are selected appropriately so that the polypeptide ofthe present invention is overproduced.

[0239] Specific media and cultivation conditions used depending on hostcells are illustrated below, but are not limited thereto.

[0240] When the hosts are bacteria, actinomycetes, yeasts, filamentousfungi, liquid media comprising the nutrient source mentioned above areappropriate. The media with pH 5 to 8 are preferably used.

[0241] When the host is E. coli, examples of preferable media are LBmedia, and M9 media (Miller et al., Exp. Mol. Genet., Cold Spring HarborLaboratory, p. 431 (1972)). Using these media, cultivation can beperformed usually at 14 to 43° C. for about 3 to 24 hours with aerationand stirring, if necessary.

[0242] When the host is Bacillus, cultivation can be performed usuallyat 30 to 40° C. for about 16 to 96 hours with aeration and stirring, ifnecessary.

[0243] When the host is yeast, examples of media are Burkholder minimalmedia (Bostian, Proc. Natl. Acad. Sci. USA, 77:4505, 1980). The pH ofthe media is preferably 5 to 8. Cultivation can be performed usually at20 to 35° C. for about 14 to 144 hours with aeration and stirring, ifnecessary.

[0244] When the host is an animal cell, examples of media are MEM mediacontaining about 5 to 20% fetal bovine serum (Science 122:501, 1952),DMEM media (Virology 8:396, 1959), RPMI1640 media (J. Am. Med. Assoc.199:519, 1967), and 199 media (Proc. Soc. Exp. Biol. Med. 73:1, 1950).The pH of the media is preferably about 6 to 8. Cultivation can beperformed usually at about 30 to 40° C. for about 15 to 72 hours withaeration and stirring, if necessary.

[0245] When the host is an insect cell, an example of media is Grace'smedia containing fetal bovine serum (Proc. Natl. Acad. Sci. USA 82:8404,1985). The pH thereof is preferably about 5 to 8. Cultivation can beperformed usually at about 20 to 40° C. for 15 to 100 hours withaeration and stirring, if necessary.

[0246] Cultivation of transformants as mentioned above, in particularanimal cells can overexpress the polypeptide of the present invention onthe surface of the cells.

[0247] The polypeptide of the present invention can be produced as asoluble polypeptide fragment such as an extracellular region fragment bypreparing the transformants as mentioned above using the DNA encodingthe extracellular region or each domain and by cultivating thetransformants to allow them to secrete the soluble polypeptide into theculture supernatant. In addition, a fusion polypeptide of the presentinvention can be prepared similarly.

[0248] Namely, a culture filtrate (supernatant) is obtained by themethod such as filtration or centrifugation of the obtained culture, andthe polypeptide or polypeptide fragment of the present invention ispurified and isolated from the culture filtrate by the usual methodcommonly used in order to purify and isolate a natural or syntheticprotein.

[0249] Examples of the isolation and purification method are a methodutilizing solubility, such as salting out and solvent precipitationmethod, a method utilizing the difference in molecular weight, such asdialysis, ultrafiltration, gel filtration, and sodium dodecylsulfate-polyacrylamide gel electrophoresis, a method utilizing charges,such as ion exchange chromatography and hydroxylapatite chromatography,a method utilizing specific affinity, such as affinity chromatography, amethod utilizing the difference in hydrophobicity, such as reverse phasehigh performance liquid chromatography, and a method utilizing thedifference in isoelectric point, such as isoelectric focusing.

[0250] When the polypeptide or a polypeptide fragment of the presentinvention exists in the periplasm or cytoplasm of culturedtransformants, first, the fungus bodies or cells are harvested by theusual method such as filtration or centrifugation and suspended inappropriate buffer. After the cell wall and/or cell membrane of thecells and so on are disrupted by the method such as lysis withsonication, lysozyme, and freeze-thawing, the membrane fractioncomprising the polypeptide of the present invention is obtained by themethod such as centrifugation or filtration. The membrane fraction issolubilized with a detergent such as Triton-X100 to obtain the crudeextract. Finally, the polypeptide or the polypeptide fragment isisolated and purified from the crude extract by the usual method asillustrated above.

[0251] The “transgenic mouse” of the present invention is a transgenicmouse wherein the DNA (cDNA or genomic DNA) prepared as mentioned aboveencoding the polypeptide of the present invention derived from animalsexcept mice (non-self polypeptide) have been integrated into itsendogenous locus of the mouse. The transgenic mouse expresses thenon-self polypeptide and secretes the polypeptide into its body.

[0252] The transgenic mouse can be prepared according to the method asusually used for producing a transgenic animal (for example, see “NewestManual of Animal Cell Experiment”, LIC press, Chapter 7, pp. 361-408,(1990)).

[0253] Specifically, for example, embryonic stem cells (ES cells)obtained from normal mouse blastocysts are transformed with anexpression vector in which the gene encoding human-derived polypeptideof the present invention (i.e., “human JTT-1 antigen”) has been operablyinserted. ES cells in which the gene encoding the human-derivedpolypeptide of the present invention has been integrated into theendogenous gene are screened by the usual method. Then, the ES cellsscreened are microinjected into a fertilized egg obtained from anothernormal mouse (blastocyst) (Proc. Natl. Acad. Sci. USA 77:7380-7384,1980; U.S. Pat. No. 4,873,191). The blastocyst is transplanted into theuterus of another normal mouse as the foster mother. Then, founder mice(progeny mice) are born from the foster mother mouse. By mating thefounder mice with normal mice, heterogeneic transgenic mice areobtained. By mating the heterogeneic transgenic mice with each other,homogenetic transgenic mice are obtained according to Mendel's laws.

[0254] Knockout mouse of the present invention is a mouse wherein theendogenous gene encoding the mouse-derived polypeptide of the presentinvention (i.e., “mouse JTT-1 antigen”) has been knocked out(inactivated). It can be prepared, for example, by positive-negativeselection method in which homologous recombination is applied (U.S. Pat.Nos. 5,464,764; 5,487,992; and 5,627,059; Proc. Natl. Acad. Sci. USA86:8932-8935, 1989; Nature 342:435-438, 1989; etc.).

[0255] The “antibody” of the present invention can be a polyclonalantibody (antiserum) or a monoclonal antibody, and preferably amonoclonal antibody.

[0256] Specifically, it is an antibody reactive to (against, which bindsto) the above-mentioned polypeptide or polypeptide fragment of thepresent invention.

[0257] The antibody of the present invention can be natural antibodiesobtained by immunizing mammals such as mice, rats, hamsters, guineapigs, and rabbits with the antigen, such as cells (natural cells, celllines, tumor cells, etc.) expressing “cell surface molecules” of thepresent invention, transformants overexpressing the polypeptide or cellsurface molecules of the present invention on the surface thereofprepared using recombinant DNA technology on the cell surface, or“polypeptide fragments” or “fusion polypeptides” of the presentinvention. The antibody of the present invention also includes chimericantibodies and humanized antibodies (CDR-grafted antibodies) that can beproduced by recombinant DNA technology, and human antibodies that can beproduced using human antibody-producing transgenic animals.

[0258] The monoclonal antibody includes those having any one isotype ofIgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.

[0259] The polyclonal antibody (antisera) or monoclonal antibody of thepresent invention can be produced by the known methods. Namely, amammal, preferably, a mouse, rat, hamster, guinea pig, rabbit, cat, dog,pig, goat, horse, or cattle, or more preferably, a mouse, rat, hamster,guinea pig, or rabbit is immunized, for example, with an antigenmentioned above with Freund's adjuvant, if necessary.

[0260] The polyclonal antibody can be obtained from the antiserumobtained from the animal so immunized. In addition, the monoclonalantibodies are produced as follows. Hybridomas are prepared from theantibody-producing cells obtained from the animal so immunized andmyeloma cells that are not capable of producing autoantibodies. Thehybridomas are cloned, and clones producing the monoclonal antibodiesshowing the specific affinity to the antigen used for immunizing themammal are screened.

[0261] Specifically, the monoclonal antibody can be produced as follows.Immunizations are performed by injecting or implanting once or severaltimes the antigen as mentioned above as an immunogen, if necessary, withFreund's adjuvant, subcutaneously, intramuscularly, intravenously,through the footpad, or intraperitoneally into a non-human mammal,specifically a mouse, rat, hamster, guinea pig, or rabbit, preferably amouse, rat, or hamster (including a transgenic animal generated so as toproduce antibodies derived from another animal such as the transgenicmouse producing human antibody mentioned below). Usually, immunizationsare performed once to four times every one to fourteen days after thefirst immunization. Antibody-producing cells are obtained from themammal so immunized in about one to five days after the lastimmunization. The frequency and interval of immunizations can beappropriately arranged depending on property of the immunogen used.Hybridomas that secrete a monoclonal antibody can be prepared by themethod of Köhler and Milstein (Nature 256:495-497, 1975) and by itsmodified method. Namely, hybridomas are prepared by fusingantibody-producing cells contained in a spleen, lymph node, bone marrow,or tonsil obtained from the non-human mammal immunized as mentionedabove, preferably a spleen, with myelomas without autoantibody-producingability, which are derived from, preferably, a mammal such as a mouse,rat, guinea pig, hamster, rabbit, or human, or more preferably, a mouse,rat, or human.

[0262] For example, mouse-derived myeloma P3/X63-AG8.653 (653),P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2),PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., orhuman-derived myeloma U-266AR1, GM15006TG-A1-2, UC729-6, CEM-AGR, D1R11,or CEM-T15 can be used as a myeloma used for the cell fusion.

[0263] Hybridoma clones producing monoclonal antibodies can be screenedby cultivating hybridomas, for example, in microtiter plates and bymeasuring the reactivity of the culture supernatant in the well in whichhybridoma growth is observed, to the immunogen used for the immunizationmentioned above, for example, by enzyme immunoassay such as RIA andELISA.

[0264] The monoclonal antibodies can be produced from hybridomas bycultivating the hybridomas in vitro or in vivo such as in the ascitesfluid of a mouse, rat, guinea pig, hamster, or rabbit, preferably amouse or rat, more preferably mouse and isolating the antibodies fromthe resulting the culture supernatant or ascites fluid of a mammal.

[0265] Cultivating hybridomas in vitro can be performed depending on theproperty of cells to be cultured, on the object of a test study, and onthe various conditions of a cultivating method, by using known nutrientmedia or any nutrient media derived from known basal media for growing,maintaining, and storing the hybridomas to produce monoclonal antibodiesin culture supernatant.

[0266] Examples of basal media are low calcium concentration media suchas Ham′F12 medium, MCDB153 medium, or low calcium concentration MEMmedium, and high calcium concentration media such as MCDB104 medium, MEMmedium, DMEM medium, RPMI1640 medium, ASF104 medium, or RD medium. Thebasal media can contain, for example, sera, hormones, cytokines, and/orvarious inorganic or organic substances depending on the objective.

[0267] Monoclonal antibodies can be isolated and purified from theculture supernatant or ascites fluid mentioned above by saturatedammonium sulfate precipitation, euglobulin precipitation method, caproicacid method, caprylic acid method, ion exchange chromatography (DEAE orDE52), affinity chromatography using anti-immunoglobulin column orprotein A column.

[0268] Preferable examples of monoclonal antibodies of the presentinvention are as follows.

[0269] (1) A monoclonal antibody reactive to a polypeptide having anamino acid sequence of SEQ ID NO: 2, a polypeptide fragment derived fromthe polypeptide, or a human-derived cell surface molecule composed ofthe polypeptide;

[0270] (2) A monoclonal antibody reactive to a polypeptide of thepresent invention, a polypeptide fragment derived from the polypeptide,or a cell surface molecule composed of the polypeptide, wherein theeffect of the monoclonal antibody on mitogen-stimulated lymphoblastcells is substantially the same as the effect of a monoclonal antibodyproduced by a hybridoma identified by an international deposit accessionNo. FERM BP-5707 on mitogen-stimulated rat lymphoblast cells; and

[0271] (3) A monoclonal antibody reactive to a polypeptide of thepresent invention, a polypeptide fragment derived from the polypeptide,or a cell surface molecule composed of the polypeptide, wherein theeffect of the monoclonal antibody on mitogen-stimulated lymphoblastcells is substantially the same as the effect of a monoclonal antibodyproduced by a hybridoma identified by an international deposit accessionNo. FERM BP-5708 on mitogen-stimulated rat lymphoblast cells.

[0272] In addition, the monoclonal antibody of the present inventionincludes that produced by the hybridoma identified by an internationaldeposit accession No. FERM BP-5707 or No. FERM BP-5708.

[0273] The “chimeric monoclonal antibody” of the present invention is amonoclonal antibody prepared by genetic engineering, and specificallymeans a chimeric antibody such as mouse/human chimeric monoclonalantibody whose variable regions are derived from immunoglobulin of annon-human mammal (mouse, rat, hamster, etc.) and whose constant regionsare derived from human immunoglobulin.

[0274] The constant region derived from human immunoglobulin has theamino acid sequence inherent in each isotype such as IgG (IgG1, IgG2,IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of therecombinant chimeric monoclonal antibody of the present invention can bethat of human immunoglobulin belonging to any isotype. Preferably, it isthe constant region of human IgG.

[0275] The chimeric monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

[0276] A mouse/human chimeric monoclonal antibody can be prepared,referring to Experimental Medicine: SUPPLEMENT, Vol. 1.6, No.10 (1988);and examined published Japanese patent application (JP-B) No. Hei3-73280. Namely, it can be prepared by operably inserting CH gene (Cgene encoding the constant region of H chain) obtained from the DNAencoding human immunoglobulin downstream of active VH genes (rearrangedVDJ gene encoding the variable region of H chain) obtained from the DNAencoding a mouse monoclonal antibody isolated from the hybridomaproducing the mouse monoclonal antibody, and CL gene (C gene encodingthe constant region of L chain) obtained from the DNA encoding humanimmunoglobulin downstream of active VL genes (rearranged VJ geneencoding the variable region of L chain) obtained from the DNA encodingthe mouse monoclonal antibody isolated from the hybridoma, into the sameor different vectors so as for them to be expressed, following bytransforming host cells with the expression vector, and then bycultivating the transformants.

[0277] Specifically, DNAs are first extracted from mouse monoclonalantibody-producing hybridomas by the usual method, digested withappropriate restriction enzymes (for example, EcoRI and HindIII),electrophoresed (using, for example, 0.7% agarose gel), and analyzed bySouthern blotting. After an electrophoresed gel is stained, for example,with ethidium bromide and photographed, the gel is given with markerpositions, washed twice with water, and soaked in 0.25 M HCl for 15minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minuteswith gently stirring. The DNAs are transferred to a filter for 4 hoursby the usual method. The filter is recovered and washed twice with2×SSC. After the filter is sufficiently dried, it is baked at 75° C. for3 hours. After baking, the filter is treated with 0.1×SSC/0.1 SDS at 65°C. for 30 minutes. Then, it is soaked in 3×SSC/0.1 SDS. The filterobtained is treated with prehybridization solution in a plastic bag at65° C. for 3 to 4 hours.

[0278] Next, ³²P-labeled probe DNA and hybridization solution are addedto the bag and reacted at 65° C. about 12 hours. After hybridization,the filter is washed under appropriate salt concentration, reactiontemperature, and time (for example, 2×SSC-0.1% SDS, room temperature, 10minutes). The filter is put into a plastic bag with a little 2×SSC, andsubjected to autoradiography after the bag is sealed.

[0279] Rearranged VDJ gene and VJ gene encoding H chain and L chain of amouse monoclonal antibody are identified by Southern blotting mentionedabove. The region comprising the identified DNA fragment is fractionedby sucrose density gradient centrifugation and inserted into a phagevector (for example, Charon 4A, Charon 28, λEMBL3, λEMBL4, etc.). E.coli (for example, LE392, NM539, etc.) is transformed with the phagevector to generate a genomic library. The genomic library is screened byplaque hybridization such as Benton-Davis method (Science 196:180-182,1977) using appropriate probes (H chain J gene, L chain (K) J gene,etc.) to obtain positive clones comprising rearranged VDJ gene or VJgene. By making the restriction map and determining the nucleotidesequence of the clones obtained, it is confirmed that genes comprisingthe desired, rearranged VH (VDJ) gene or VL (VJ) gene are obtained.

[0280] Separately, human CH gene and human CL gene used forchimerization are isolated. For example, when a chimeric antibody withhuman IgG1 is produced, Cγ1 gene as a CH gene, and Cκ gene as a CL gene,are isolated. These genes can be isolated from human genomic librarywith mouse Cγ1 gene and mouse Cκ gene, corresponding to human Cγ1 geneand human Cκ gene, respectively, as probes, taking advantage of highhomology between the nucleotide sequences of mouse immunoglobulin geneand that of human immunoglobulin gene.

[0281] Specifically, DNA fragments comprising human Cκ gene and anenhancer region are isolated from human 80 Charon 4A HaeIII-AluI genomiclibrary (Cell 15:1157-1174, 1978), for example, with a 3 kbHindIII-BamHI fragment of clone Ig146 (Proc. Natl. Acad. Sci. USA75:4709-4713, 1978) and a 6.8 kb EcoRI fragment of clone MEP10 (Proc.Natl. Acad. Sci. USA 78:474-478, 1981) as probes. In addition, forexample, after human fetal hepatocyte DNA is digested with HindIII andfractioned by agarose gel electrophoresis, a 5.9 kb fragment is insertedinto X788 and then human Cγ1 gene is isolated with the probes mentionedabove.

[0282] Using mouse VH gene, mouse VL gene, human CH gene, and human CLgene so obtained, and taking promoter region and enhancer region intoconsideration, human CH gene is inserted downstream mouse VH gene andhuman CL gene is inserted downstream mouse VL gene into an expressionvector such as pSV2gpt or pSV2neo with appropriate restriction enzymesand DNA ligase by the usual method. In this case, chimeric genes ofmouse VH gene/human CH gene and mouse VL gene/human CL gene can berespectively inserted in the same expression vector or in differentexpression vectors.

[0283] Chimeric gene-inserted expression vector(s) thus prepared areintroduced into myelomas that do not produce antibodies, for example,P3×63∘Ag8∘653 cells or SP210 cells by protoplast fusion method,DEAE-dextran method, calcium phosphate method, or electroporationmethod. The transformants are screened by cultivating in mediacontaining a drug corresponding to the drug resistance gene insertedinto the expression vector and, then, cells producing desired chimericmonoclonal antibodies are obtained.

[0284] Desired chimeric monoclonal antibodies are obtained from theculture supernatant of antibody-producing cells thus screened.

[0285] The “humanized monoclonal antibody (CDR-grafted antibody)” of thepresent invention is a monoclonal antibody prepared by geneticengineering and specifically means a humanized monoclonal antibodywherein a portion or the whole of the complementarity determiningregions of the hypervariable region are derived from the complementaritydetermining regions of the hypervariable region from a monoclonalantibody of an non-human mammal (mouse, rat, hamster, etc.), theframework regions of the variable region are derived from the frameworkregions of the variable region from human immunoglobulin, and theconstant region is derived from human a constant region fromimmunoglobulin.

[0286] The complementarity determining regions of the hypervariableregion exists in the hypervariable region in the variable region of anantibody and means three regions which directly and complementary bindsto an antigen (complementarity-determining residues, CDR1, CDR2, andCDR3). The framework regions of the variable region means fourcomparatively conserved regions lying upstream, downstream or betweenthe three complementarity determining regions (framework region, FR1,FR2, FR3, and FR4).

[0287] In other words, a humanized monoclonal antibody means that inwhich the whole region except a portion or the whole of thecomplementarity determining regions of the hypervariable region of anonhuman mammal-derived monoclonal antibody have been replaced withtheir corresponding regions derived from human immunoglobulin.

[0288] The constant region derived from human immunoglobulin has theamino acid sequence inherent in each isotype such as IgG (IgG1, IgG2,IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a humanizedmonoclonal antibody in the present invention can be that from humanimmunoglobulin belonging to any isotype. Preferably, it is the constantregion of human IgG. The framework regions of the constant regionderived from human immunoglobulin are not particularly limited.

[0289] The humanized monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

[0290] For example, a recombinant humanized monoclonal antibody derivedfrom mouse monoclonal antibody can be prepared by genetic engineering,referring to unexamined Japanese patent publication (JP-WA) No. Hei4-506458 and unexamined Japanese patent publication (JP-A) No. Sho62-296890. Namely, at least one mouse H chain CDR gene and at least onemouse L chain CDR gene corresponding to the mouse H chain CDR gene areisolated from hybridomas producing mouse monoclonal antibody, and humanH chain gene encoding the whole regions except human H chain CDRcorresponding to mouse H chain CDR mentioned above and human L chaingene encoding the whole region except human L chain CDR correspond tomouse L chain CDR mentioned above are isolated from human immunoglobulingenes.

[0291] The mouse H chain CDR gene(s) and the human H chain gene(s) soisolated are operably inserted into an appropriate vector so that theycan be expressed. Similarly, the mouse L chain CDR gene(s) and the humanL chain gene(s) are operably inserted into another appropriate vector sothat they can be expressed. Alternatively, the mouse H chain CDRgene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human Lchain gene(s) can be operably inserted into the same expression vectorso that they can be expressed. Host cells are transformed with theexpression vector thus prepared to obtain transformants producinghumanized monoclonal antibody. By cultivating the transformants, desiredhumanized monoclonal antibody is obtained from culture supernatant.

[0292] The “human monoclonal antibody” of the present invention isimmunoglobulin in which the entire regions comprising the variable andconstant region of H chain, and the variable and constant region of Lchain constituting immunoglobulin are derived from the gene encodinghuman immunoglobulin.

[0293] The human antibody can be produced in the same way as theproduction method of polyclonal or monoclonal antibodies mentioned aboveby immunizing, with an antigen, a transgenic animal which for example,at least human immunoglobulin gene(s) have been integrated into thelocus of a non-human mammal such as a mouse by the usual method.

[0294] For example, a transgenic mouse producing human antibodies isprepared by the methods described in Nature Genetics 7:13-21, 1994;Nature Genetics 15:146-156, 1997; JP-WA Nos. Hei 4-504365 and Hei7-509137; Nikkei Science 6:40-50, 1995; International patent publicationNo. WO94/25585; Nature 368:856-859, 1994; and JP-WA No. Hei 6-500233.

[0295] In addition, recently developed technique for producing ahuman-derived protein from the milk of a transgenic cow or pig can alsobe applied (Nikkei Science, pp. 78-84, April, 1997).

[0296] The “portion of an antibody” used in the present invention meansa partial region of the monoclonal antibody as mentioned above, andspecifically, means F(ab′)₂, Fab′, Fab, Fv (variable fragment ofantibody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domainantibody) (Exp. Opin. Ther. Patents 6:441-456, 1996).

[0297] “F(ab′)₂” and “Fab′” can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andmeans an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds existing between the hinge regions in each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds existing between the hinge regions in each of the two H chains togenerate two homologous antibody fragments in which an L chain composedof VL (L chain variable region) and CL (L chain constant region), and anH chain fragment composed of VH (H chain variable region) and CHγ1 (γ1region in the constant region of H chain) are connected at their Cterminal regions through a disulfide bond. Each of such two homologousantibody fragments is called Fab′. Pepsin also cleaves IgG downstream ofthe disulfide bonds existing between the hinge regions in each of thetwo H chains to generate an antibody fragment slightly larger than thefragment in which the two above-mentioned Fab′ are connected at thehinge region. This antibody fragment is called F(ab′)₂.

[0298] The “pharmaceutical composition” of the present inventioncomprises any one of the “polypeptides” of the present invention asdefined above; “homodimer molecule”, “polypeptide fragment”, “fusionpolypeptide” comprising the polypeptide; “homodimer molecule” comprisingthe fusion polypeptides, “antibody”, or “portion of an antibody”; and apharmaceutically acceptable carrier.

[0299] The “pharmaceutically acceptable carrier” includes a excipient, adiluent, an expander, a decomposition agent, a stabilizer, apreservative, a buffer, an emulsifier, an aromatic, a colorant, asweetener, a viscosity increasing agent, a flavor, a solubilityincreasing agent, or other additives. Using one or more of suchcarriers, a pharmaceutical composition can be formulated into tablets,pills, powders, granules, injections, solutions, capsules, troches,elixirs, suspensions, emulsions, or syrups. The pharmaceuticalcomposition can be administered orally or parenterally. Other forms forparenteral administration include a solution for external application,suppository for rectal administration, and pessary, prescribed by theusual method, which comprises one or more active ingredient.

[0300] The dosage can vary depending on the age, sex, weight, andsymptom of a patient, effect of treatment, administration route, periodof treatment, or the kind of active ingredient (polypeptide or antibodymentioned above) contained in the pharmaceutical composition. Usually,the pharmaceutical composition can be administered to an adult in a doseof 10 μg to 1000 mg (or 10 μg to 500 mg) per one administration.Depending on various conditions, the dosage less than that mentionedabove may be sufficient in some cases, and the dosage more than thatmentioned above may be necessary in other cases.

[0301] In particular, the injection can be produced by dissolving orsuspending the antibody in a non-toxic, pharmaceutically acceptablecarrier such as physiological saline or commercially available distilledwater for injection with adjusting a concentration to 0.1 μg antibody/mlcarrier to 10 mg antibody/ml carrier. The injection thus produced can beadministered to a human patient in need of treatment in a dose of 1 μgto 100 mg/kg body weight, preferably 50 μg to 50 mg/kg body weight onceor more times a day. Examples of administration route are medicallyappropriate administration routes such as intravenous injection,subcutaneous injection, intradermal injection, intramuscular injection,or intraperitoneal injection, preferably intravenous injection.

[0302] The injection can also be prepared into a non-aqueous diluent(for example, propylene glycol, polyethylene glycol, vegetable oil suchas olive oil, and alcohol such as ethanol), suspension, or emulsion.

[0303] The injection can be sterilized by filtration with abacteria-non-penetrated filter, by mixing bactericide, or byirradiation. The injection can be produced in the form that is preparedupon use. Namely, it is freeze-dried to be a sterile solid composition,and can be dissolved in sterile distilled water for injection or anothersolvent before use.

[0304] The pharmaceutical composition of the present invention can beapplied to treating or preventing various autoimmune diseases, allergicdiseases, or inflammatory diseases caused by the activation oflymphocytes such as T cells and the regulation of activated lymphocytefunctions. Examples of the diseases are rheumatoid arthritis, multiplesclerosis, autoimmune thyroiditis, allergic contact dermatitis, chronicinflammatory dermatosis such as lichen planus, systemic lupuserythematosus, insulin dependent diabetes mellitus, and psoriasis.

[0305] The therapeutic effect of the pharmaceutical composition of thepresent invention for symptom of various diseases can be tested by theusual method by administering it to an known disease model animal.

[0306] Examples of the model include (1) a (NZB/NZW)F1 mouse, a modelfor human systemic lupus erythematosus (SLE) (Science 125:1225-1227,1994); (2) experimental allergic encephalomyelitis (EAE), a model formultiple sclerosis (MS) (J. Clin. Invest. 95:2783-2789, 1995); (3) anNOD (non-obese diabetes) mouse, a model for insulin dependent diabetesmellitus (IDDM) (J. Exp. Med. 181:1145-1155, 1995); (4) rat nephritismodel by renal glomerulus basement membrane immunity, Goodpasture'snephritis model (Eur. J. Immunol. 24:1249-1254, 1994); and (5) a DBA/1mouse, a model for human rheumatoid arthritis (Eur. J. Immunol.26:2320-2328, 1996).

BRIEF DESCRIPTION OF THE DRAWINGS

[0307]FIG. 1 are micrographs showing the state of aggregation of FTL435cells induced by “JTT-1 antibody” and the state of inhibition of thecell aggregation by “JTT.2 antibody.”

[0308] Subfigure (a) shows the state of the cells in the absence of anyhybridoma supernatant, subfigure (b) shows the state of cell aggregationinduced by “JTT-1 antibody,” subfigure (c) shows the state of the cellaggregation in the presence of “anti-ICAM-1 antibody” together with“JTT-1 antibody,” and subfigure (d) shows the state of the cellaggregation in the presence of “JTT-2 antibody” together with “JTT-1antibody.”

[0309]FIG. 2 are micrographs showing the state of aggregation of FTL435cells and rat activated lymphoblasts induced by “JTT-1 antibody” and thestate of inhibition of the cell aggregation by “JTT.2 antibody.”

[0310] Subfigure (a) shows the state of FTL435 cells in the absence ofany antibody, subfigure (b) shows the state of FTL435 cells in thepresence of PMA, subfigure (c) shows the state of FTL435 cells in thepresence of “JTT-1 antibody,” subfigure (d) shows the state of FTL435cells in the presence of anti-LFA-1 antibody together with “JTT-1antibody,” subfigure (e) shows the state of FTL435 cells in the presenceof anti-CD18 antibody together with “JTT-1 antibody,” subfigure (f)shows the state of FTL435 cells in the presence of anti-ICAM-1 antibodytogether with “JTT-1 antibody,” subfigure (g) shows the state ofactivated lymphoblasts in the absence of any antibody, subfigure (h)shows the state of activated lymphoblasts in the presence of PMA,subfigure (i) shows the state of activated lymphoblasts in the presenceof “JTT-1 antibody,” subfigure (j) shows the state of activatedlymphoblasts in the presence of anti-LFA-1 antibody together with “JTT-1antibody,” subfigure (k) shows the state of activated lymphoblasts inthe presence of anti-CD18 antibody together with “JTT-1 antibody,” andsubfigure (1) shows the state of activated lymphoblasts in the presenceof anti-ICAM-1 antibody together with “JTT-1 antibody.”

[0311]FIG. 3 shows the expression state of “JTT-1 antigen” and “JTT-2antigen” in various cells measured with a flow-cytometer.

[0312]FIG. 4 shows the expression state of “JTT-1 antigen” in variouslymphocytic cells measured with a flow-cytometer.

[0313]FIG. 5 is a photograph showing electrophoretogram of “JTT-1antigen” analyzed by SDS-PAGE.

[0314]FIG. 6 are micrographs showing the state of adhesion of ratthymocytes to the microtiter plate coated with purified “JTT-1 antigen,”where the adhesion is induced in the presence of “JTT-1 antibody,” andthe state of inhibition of the cell adhesion by “JTT-2 antibody.”

[0315] Subfigure (a) shows the state of adhesion of the cells to theplate which has not been coated with “JTT-1 antigen,” subfigure (b)shows the state of adhesion of the cells to the plate coated with “JTT-1antigen” in the absence of any antibody, subfigure (c) shows the stateof adhesion of the cells to the plate coated with “JTT-1 antigen” in thepresence of the Fab fragments of “JTT-1 antibody,” and subfigure (d)shows the state of adhesion of the cells to the plate coated with “JTT-1antigen” in the presence of “JTT-2 antibody” together with the Fabfragments of “JTT-1 antibody.”

[0316]FIG. 7 shows the relative cell number of thymocytes adhering tothe plate coated with purified “JTT-1 antigen” measured in terms offluorescence intensity.

[0317] “Ag(−)” shows the relative cell number in the plate which has notbeen coated with “JTT-1 antigen,” “Ag(+)” shows the relative cell numberin the plate coated with “JTT-1 antigen” in the absence of any antibody,“Ag(+)+JTT-1 Fab” shows the relative cell number in the plate coatedwith “JTT-1 antigen” in the presence of the Fab fragments of “JTT-1antibody”, and “Ag(+)+JTT-1 Fab+JTT-2 “1 shows the relative cell numberin the plate coated with “JTT-1 antigen” in the presence of “JTT-2antibody” together with the Fab fragments of “JTT-1 antibody.”

[0318]FIG. 8 shows the expression state of “rat JTT-1 antigen” and “ratJTT-2 antigen” in COS cells transformed with cDNA encoding “rat JTT-1antigen” with a flow-cytometer.

[0319]FIG. 9 shows the structural characteristics of amino acid sequenceof “JTT-1 antigen” revealed by hydropathy plot analysis.

[0320]FIG. 10 shows the homology among amino acid sequences of human,rat, and mouse “JTT-1 antigen” and “rat JTT-1 antigen” mutant.

[0321]FIG. 11 shows the homology among amino acid sequences andconservation state of motifs in “human JTT-1 antigen,” “human CD28molecule”, and “human CTLA-4 molecule.”

[0322]FIG. 12 schematically shows the protein secondary structure of,and their similarity among “human JTT-1 antigen,” “human CD28 molecule,”and “human CTLA-4 molecule.”

[0323]FIG. 13 schematically shows the structure of the genomic DNAencoding “mouse JTT-1 antigen.”

[0324]FIG. 14 shows the difference in amino acid sequences between “ratJTT-1 antigen” and its alternative splicing mutant.

[0325]FIG. 15 shows the degree of the growth of human peripheral bloodlymphocytes induced by the monoclonal antibody against “human JTT-1antigen,” where the degree of the growth was measured by [³H] thymidineuptake.

[0326] The ordinate shows the amount of uptake (dpm) of [³H] thymidineinto the cells.

[0327]FIG. 16 shows the therapeutic effect of the monoclonal antibodyagainst “JTT-1 antigen” on experimental allergic encephalomyelitis (EAE)in a disease model rat.

[0328] The ordinate shows the scored degree of disease symptom, and theabscissa shows the days after immunization for induction of EAE.

[0329]FIG. 17 shows the therapeutic effect of the monoclonal antibodyagainst “JTT-1 antigen” on glomerulonephritis in a disease model rat.

[0330] The ordinate shows the amount of urinary excretion of proteins,and the abscissa shows the time course (week) after immunization forinduction of glomerulonephritis

[0331]FIG. 18 shows a column histogram in purification of fusionpolypeptide between “rat JTT-1 antigen” extracellular region and humanIgFc (rJTT-1-IgFc) with protein A Sepharose column.

[0332]FIG. 19 is a photograph showing electrophoretogram of rJTT-1-IgFcanalyzed by SDS-PAGE.

[0333]FIG. 20 shows a column histogram in purification of fusionpolypeptide between “human JTT-1 antigen” extracellular region and humanIgFc (hJTT-1-IgFc) with protein A Sepharose column.

[0334]FIG. 21 is a photograph showing electrophoretogram of hJTT-1-IgFcanalyzed by SDS-PAGE.

[0335]FIG. 22 schematically shows the structure of the gene transfer(targeting) vector used for preparation of a transgenic mouse into whichthe cDNA encoding “rat JTT-1 antigen” has been introduced.

BEST MODE FOR IMPLEMENTING THE INVENTION

[0336] The present inventions are described in more detail withreference to Examples below, but are not to be construed to be limitedthereto.

EXAMPLE 1 Preparation of Monoclonal Antibodies

[0337] Antibody-producing hybridomas were prepared according to themethod of Köhler et al. (Omori et al., Blood, 81:101-111, 1993), andmonoclonal antibodies were prepared according to the method of Kannagiet al. (Handbook of Experimental Immunology, 4:117.21-117.21, 1986).

[0338] First, rat thymoma cell line FTL435 cells were administered as animmunizing antigen to BALB/c mice into their footpad in an amount of 10⁷cells/mouse at intervals of 0, 7, 14, and 28 days. The mixture of theantigen with Freund's complete adjuvant was administered only in thefirst immunization. Two days after the last immunization, the lymphnodes of the mice were taken out and fused with mouse myeloma cells PAI(JCR No. B0113; Stocker et al., Res. Disclosure, 217:155, 1982) by theusual method to obtain many hybridomas producing monoclonal antibodies.

Example 2 Screening of Hybridomas and Characterization of MonoclonalAntibodies

[0339] The hybridomas prepared in Example 1 were screened by analyzingthe effect of the antibodies produced in the culture supernatant of thehybridomas on FTL435 cells, which were used as the immunogen. FTL435cells (5×10⁶ cells/ml, 0.1 ml) were seeded into each well of a 96-wellmicrotiter plate and cultivated at 37° C. for an hour in the presence ofculture supernatant of each hybridoma (10 μg/ml each). The resultsobtained for hybridoma clones “JTT-1” and “JTT-2” are shown in FIG. 1and FIG. 2.

[0340] It was revealed that a monoclonal antibody produced by hybridomaclone “JTT-1” (“JTT-1 antibody”) strongly agglutinated FTL435 cells(FIG. 1(b) and FIG. 2(c)) and that addition of “JTT-2 antibody” stronglyinhibited the aggregation of FTL435 cells induced by “JTT-1 antibody”stimulation (FIG. 1(d)). The assays, in which no hybridoma supernatantwas added, were used as controls (FIG. 1(a) and FIG. 2(a)).

[0341] In order to determine whether the aggregation of FTL435 cellsinduced by “JTT-1 antibody” stimulation was caused by the cell adhesionbetween intercellular adhesion molecule-1 (ICAM-1) and lymphocytefunction-associated antigen-1 (LFA-1), which is a representative knownpathway of cell adhesion, FTL435 cells were cultivated at 37° C. for anhour in the presence of anti-rat ICAM-1 antibody 1A29 (10 μg/ml; IgG1)or anti-rat LFA-1 antibody (10 μg/ml; IgG2a) together with “JTT-1antibody.”

[0342] The aggregation of FTL435 cells by “JTT-1 antibody” stimulationwas inhibited by neither anti-ICAM-1 antibody nor anti-LFA-1 antibody(anti-ICAM-1 antibody, FIG. 1(c) and FIG. 2(f); anti-LFA-1 antibody,FIG. 2(d)).

[0343] In order to further analyze the cell agglutination ability of“JTT-1 antibody,” the ability to agglutinate rat lymphoblast cellsactivated with concanavalin A stimulation was analyzed in the samemanner as mentioned above. The results are shown in FIG. 2.

[0344] Similar to the effect on FTL435 cells, the aggregation ofactivated lymphoblast cells was induced by “JTT-1 antibody” stimulation(FIG. 2(i)). The aggregation of activated lymphoblast cells by “JTT-1antibody” stimulation was mostly inhibited by anti-LFA-1 antibody (FIG.2(j)) and anti-ICAM-1 antibody (FIG. 2(l)). (However, partialaggregation occurred.)

[0345] As understood from the control assay (FIG. 2(g)), in which noantibody was added, activated lymphocytes such as activated lymphoblastsshowed no aggregation through cell adhesion unless they receive thestimulation with phorbol myristate acetate (PMA, which activates LFA-1)(FIG. 2(h)) or “JTT-1 antibody” (FIG. 2(i)). Therefore, the fact thatanti-LFA-1 antibody partially inhibited cell aggregation by “JTT-1antibody” stimulation indicates that LFA-1 in activated lymphoblastcells was activated by “JTT-1 antibody” stimulation. This also indicatesthat molecules recognized by “JTT-1 antibody” are involved in somesignal transmission.

[0346] Hybridoma clones “JTT-1” and “JTT-2” have been deposited underthe Budapest Treaty with international depository authority, NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry,Japan (1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) since Oct. 11, 1996with an international accession Nos. FERM BP-5707 and FERM BP-5708,respectively.

[0347] Analysis using mouse monoclonal antibody isotype identificationkit (Amersham) determined that the isotype of monoclonal antibodiesproduced from each hybridoma (JTT-1 antibody and JTT-2 antibody) wereboth IgG1.

Example 3 Reactivity of “JTT-1 Antibody” and “JTT-2 Antibody” to VariousCells

[0348] In order to analyze the expression pattern of moleculesrecognized by “JTT-1 antibody” and “JTT-2 antibody” in various cells,the reactivities of the antibodies to various cells were examined.Molecules recognized by “JTT-1 antibody” or “JTT-2 antibody” aredesignated “JTT-1 antigen” or “JTT-2 antigen”, respectively.

[0349] A five- to ten-week-old Wistar rat (150 to 250 g) was killed byanesthesia with diethyl ether. The thymus and spleen were taken out ofits chest and abdomen, respectively, by celiotomy, and homogenized toprepare cell suspension. The resulting spleen cells were cultivated inRPMI1640 medium containing 2 μg/ml concanavalin A and 10% FCS at 37° C.for 3 days to prepare activated lymphoblasts.

[0350] FTL435 cells, thymocytes, spleen cells, and activatedlymphoblasts (5×10 ⁵ cells each) were reacted with “JTT-1 antibody” or“JTT-2 antibody” and then with FITC-labeled anti-mouse IgG (Cappel). Thefluorescence intensity of the stained cells was measured withEPICS-Elite flow cytometer.

[0351] The results are shown in FIG. 3. In FTL435 cells, the strongexpression of each “JTT-1 antigen” and “JTT-2 antigen” was observed.While the antigens were expressed in thymocytes, they were expressedonly a little in spleen cells. However, in activated lymphoblastsobtained by simulating spleen cells with concanavalin A, “JTT-1 antigen”and “JTT-2 antigen” were strongly expressed. In addition, in each kindof cells, the expression pattern of “JTT-1 antigen” and “JTT-2 antigen”coincided with each other. These results indicate that “JTT-1 antigen”and “JTT-2 antigen” are the same molecules.

Example 4 Reactivity of “JTT-1 Antibody” to Various Lymphocytic Cells

[0352] In order to analyze the expression pattern of molecules (“JTT-1antigen”) recognized by “JTT-1 antibody” in various lymphocytic cells,the reactivity of “JTT-1 antibody” to lymph nodes, T lymphoblastsderived from spleen, and B lymphoblasts derived from spleen of two kindsof rats (Wistar rat and F344 rat) was analyzed.

[0353] A five- to ten-week-old Wistar rat and F344 rat (150 to 250 g)were killed by anesthesia with diethyl ether. The lymph nodes and spleenwere taken out of each rat by celiotomy, and homogenized to prepare cellsuspension. The resulting cell suspension from spleen was cultivated inRPMI1640 medium containing 2 μg/ml concanavalin A (ConA) and 10% FCS at37° C. for 3 days. Activated T lymphoblasts and activated B lymphoblastswere obtained from each rat after 1-day and 3-day cultivation. Inaddition, spleen-derived T lymphoblasts and B lymphoblasts obtainedbefore lymph node cells and ConA were added were used as controls.

[0354] Each cells (5×10⁵ cells each) were reacted with biotin-labeledanti-rat T cell antibody or biotin-labeled anti-rat B cell antibody (10μg/ml, Seikagaku Corporation), and subsequently withphycoerythrin-labeled streptavidin. The cells were then reacted with 10μg/ml FITC-labeled “JTT-1 antibody.” The fluorescence intensity of thestained cells was measured with EPICS-Elite flow cytometer.

[0355] The results are shown in FIG. 4. In both activated T lymphoblastsand activated B lymphoblasts from Wistar rat and F344 rat, the strongexpression of “JTT-1 antigen” was observed from day 1 of activation withConA stimulation. In addition, the expression pattern of “JTT-1 antigen”in each kind of cells almost perfectly coincided with each other.

Example 5 Characterization of “JTT-1 Antigen” and “JTT-2 Antigen” byImmunoprecipitation

[0356] “JTT1 antigen” and “JTT-2 antigen” were characterized byimmunoprecipitation using FTL435 cells.

[0357] (1) Preparation of Biotinylated Soluble Cell Surface Molecules

[0358] FTL435 cells were washed with PBS, suspended in physiologicalsaline containing 100 μg/ml NHS-biotin and 0.1 M HEPES (pH 8.0) toadjust 1×10′ cells/ml, and incubated at room temperature for 40 minutes.The cells were washed three times with PBS, lysis buffer (1% NP-40, 10mM Tris-HCl (pH 7.4), 0.15 M NaCl) was added thereto to adjust 5×10⁷cells/ml, and the mixture was allowed to react at 4° C. for 30 minutesto lyse the cells. The cell lysate obtained was centrifuged, and thesupernatant comprising biotinylated soluble cell surface molecules wasstored at −80° C.

[0359] (2) Immunoprecipitation and SDS-PAGE Analysis

[0360] The purified sample of “JTT-1 antibody” purified by the usualmethod from the culture supernatant of the hybridoma clone “JTT-1”prepared in Example 1 was mixed with protein G-Sepharose beads to adjust2 mg/ml, and allowed to react at 4° C. for an hour to bind the antibodywith the beads. After the beads were washed, 500 μl of the biotinylatedFTL435 cell lysate was added to 10 μl of the beads, and the mixture wasallowed to react at 40° C. for 2 hours. After the beads were washed withlysis buffer three times, 50 μl of glycanase buffer (sodium phosphatebuffer (pH 7.0) containing 0.15% SDS) was added to the beads, and themixture was boiled to elute the bound molecules trapped by theantibody-bound beads. 1.25% NP-40 and 20 U/ml N-glycanase were added toa fraction of the sample so eluted, and the mixture was allowed to reactovernight to digest N-linked sugar chains.

[0361] An equal volume of sample buffer (Enprotech) for SDS-PAGE wasadded to 5 μl of the eluted sample in the presence or absence of2-mercaptoethanol, and the mixture was boiled. After electrophoresis,the gel was transferred to a PVDF membrane. The membrane was blockedwith 3% BSAPBS and reacted with peroxidase-labeled streptavidin todetect biotinylated soluble cell surface molecules trapped by “JTT-1antibody” with ECL system (Amersham) as described in the manual.

[0362] The results are shown in FIG. 5. The “JTT-1 antibody”-recognizedmolecule (“JTT-1 antigen”) on FTL435 cells showed the molecular weightof about 47 kD under the non-reduced conditions (“(−)” in FIG. 5) andabout 24 kD and 28 kD under the reduced conditions (“(+)” in FIG. 5). Asthe result of digestion of N-linked sugar chains (”+N−gly” in FIG. 5),“JTT-1 antigen” was converged on a single band of about 36 kD under thenon-reduced conditions and about 20 kD under the reduced conditions.These results suggest that “JTT-1 antigen” forms a dimer in which thesame core proteins have different sugar chains. Completely the sameresults were obtained in the experiment performed as mentioned aboveusing “JTT-2 antibody.” Considering these results together with theresults of Example 3 and Example 7 below, “JTT-1 antigen” (moleculerecognized by “JTT-1 antibody”) and “JTT-2 antigen” (molecule recognizedby “JTT2 antibody”) have been thought to be identical to each other.

Example 6 Adhesion Experiment of Rat Thymocytes to Purified “JTT-1Antigen” and N Terminal Amino Acid Analysis

[0363] The following experiments were performed to analyze whether themolecule that “JTT-1 antibody” recognizes (“JTT-1 antigen”) functions asan adhesion molecule. N-terminal amino acid analysis was also performed.

[0364] (1) Preparation of “JTT-1 Antibody”-Affinity Column

[0365] The purified sample (2 mg in 2 ml) of “JTT-1 antibody” purifiedby the usual method from the culture supernatant of the hybridoma clone“JTT-1” prepared in Example 1 was mixed with 1 ml of protein G-Sepharoseresin, and the mixture was allowed to react at 40° C. for an hour. Theresin was washed three times with 200 mM triethanolamine (pH 8.2). Theresin was then incubated in triethanolamine (pH 8.2) containing 10 mMdimethyl pimelimidate (DMP) at room temperature for an hour tocovalently bind “JTT-1 antibody” to the resin.

[0366] (2) Purification of “JTT-1 Antigen”

[0367] FTL435 cells were cultivated in RPMI1640 medium containing 10%FCS. The cells were harvested by centrifugation to obtain a pellet andwashed with PBS three times. Lysis buffer (1% NP-40, 10 mM Tris-HCl (pH7.4), 0.15 M NaCl) was added to the washed pellet to adjust 5×10⁷cells/ml, and the mixture was allowed to react at 4° C. for 30 minutesto lyse the cells. The cell lysate obtained was centrifuged, and thesupernatant containing soluble cell surface molecules was stored at−800° C.

[0368] The lysate (400 ml) was loaded onto “JTT-1 antibody”-affinitycolumn. After the column was washed with 50 ml of the lysis buffer and20 ml of PBS, “JTT-1 antigen” was eluted with 0.2 M glycine buffer (pH2.8). 1 M Tris buffer was added to the “JTT-1 antigen” so eluted forneutralization. “JTT-1 antigen” obtained was stored at −80° C.

[0369] (3) Determination of N Terminal Amino Acid Sequence

[0370] After the purified “JTT-1 antigen” was subjected to SDS-PAGE, theN-terminal amino acid sequence was determined by the usual method. Theresult revealed that “JTT-1 antigen” contained an amino acid sequenceGlu-Leu-Asn-AspLeu-Ala-Asn-His-Arg.

[0371] (4) Adhesion Experiment

[0372] A five- to ten-week-old Wistar rat (150 to 250 g) was killed byanesthesia with diethyl ether. The thymus was taken out of its chest byceliotomy and homogenized to prepare thymocyte suspension. 10μ,7′-bis(carboxyethyl)carboxyfluorescein tetraacetoxy-methyl ester(BCECF-AM; Molecular Probes) was added to the suspension, and themixture was incubated at 37° C. for 30 minutes to fluorescently labelthe thymocytes. The cells were washed with PBS and suspended in RPMI1640medium containing 10% FCS to adjust 2×10⁷ cells/ml.

[0373] The purified “JTT-1 antigen” obtained in (2) was coated on a96-well ELISA plate at the concentration of 10 μl/well overnight. Afterthe plate was washed with PBS, 200 μl/well of PBS containing 3% BSA wasadded to the plate, and blocking was performed for 2 hours. After theplate was washed with PBS, (1) only fluorescence-labeled thymocytes(2×10⁷ cells/ml, 0.1 ml); (2) fluorescence-labeled thymocytes (sameconcentration) and “JTT-1 antibody” Fab fragments prepared by the usualmethod (5μ “JTT-1 antibody” Fab fragments (same concentration), and“JTT-2 antibody” (10μ

[0374] ° C. for an hour. In order to remove unbound cells, each well waswashed once with RPMI1640 medium containing 10% FCS. Each well wasobserved with light microscope. Then, 100 μl of 0.1% NP-40 was added toeach well, and the cells adhered to the plate were lysed. The relativecell number of fluorescence-labeled thymocytes adhered to each well wascounted by measuring the fluorescence intensity at 538 nm (excited at485 nm) with Fluoroscan II Microplate Fluorometer (Flow Laboratories).The assay in which a plate was not coated with purified “JTT-1 antigen”was used as a control.

[0375] The results of light microscopy observation are shown in FIG. 6.

[0376] Thymocytes significantly adhered to purified “JTT-1 antigen” onlyin the presence of “JTT-1 antibody” Fab fragments (FIG. 6(c)). Theadhesion was significantly inhibited by “JTT-2 antibody” (FIG. 6(d)).

[0377]FIG. 7 shows the relative cell number of thymocytes adhered to“JTT-1 antigen” coated on each well in terms of fluorescent intensity.

[0378] From these results, it was revealed that “JTT-1 antigen”functions as an adhesion molecule.

Example 7 Cloning of cDNA Encoding Rat “JTT-1 Antigen”

[0379] 1. Preparation of cDNA Library

[0380] 1-(1) Extraction of Poly(A)⁺ RNA From ConA-Stimulated RatLymphoblasts

[0381] ConA-stimulated lymphoblasts (ConA blast) derived from rat spleen(about 1×10⁶ cells/ml) were centrifuged (2,000×g) at 4° C. for 5minutes. The precipitated cells were suspended with ISOGEN (Nippon Gene)and extracted with chloroform with shaking to collect the supernatant.After isopropanol was added to the obtained supernatant, the mixture wasallowed to stand at room temperature for 10 minutes and centrifuged at12,000×g at 4° C. for 10 minutes to precipitate RNA. The precipitatedRNA was washed with ethanol and dissolved in TE buffer. Poly(A)⁺ RNA waspurified from the total RNA so obtained with “mRNA Purification Kit”(Pharmacia).

[0382] 1-(2) Preparation of cDNA

[0383] With 5 μg of the poly(A)⁺ RNA prepared above as a template, cDNAwas synthesized with “Time Saver cDNA Synthesis Kit” (Pharmacia). “OligodT primer” (Pharmacia) having NotI site was used to increase theefficiency of screening. EcoRI adapter was added, and digestion withNotI was performed to obtain cDNA with unidirectionality. Sizefractionation was then performed with Spun Column (Pharmacia).

[0384] 1-(3) Insertion Into a Vector

[0385] The obtained cDNA having EcoRI- and NotI-ends was ligated withpME18S (Hara et al., EMBO J., 11:1875-1884, 1992) digested with EcoRIand NotI. “DNA Ligation Kit” (Takara Shuzo) was used for the ligation.E. coli DH5 cells (Toyobo) were transformed with the reaction product soobtained. Transformants were cultivated until O.D. value (at 600 nm)reached 0.6 and harvested to recover plasmid DNAs with a library.QUIAGEN-Tip (QUIAGEN) was used for purification of plasmid DNAs.

[0386] 2. Screening of cDNA Library

[0387] Screening was performed according to panning method (Seed et al.,Proc. Natl. Acad. Sci. USA, 84:3365-3369, 1987).

[0388] 2-(1) Gene Transfer into COS Cells

[0389] The library so obtained was introduced into COS7 cells byelectroporation (Potter et al., Proc. Natl. Acad. Sci. USA,85:2288-2292). The transformants were cultivated for 60 hours afterintroduction, the supernatant was removed, and the pellet was washedwith PBS three times. After the pellet was treated with PBS (containing0.5 mM EDTA) at 37° C. for 30 minutes, the cells were removed bypipetting. Only living cells were then collected with “Lymphprep”(NYCOMED).

[0390] 2-(2) Concentration of Gene-Expressing Cells by Panning

[0391] The living cells obtained above were suspended in PBS (containing5% FCS and 0.5 mM EDTA). The cell suspension was transferred to aculture dish coated with “JTT-1 antibody” and incubated at roomtemperature for 3 hours. After cells not binding to the culture dishwere removed and the culture dish was washed with PBS three times,plasmid DNAs were collected from the cells binding to the culture dishby Hirt method (Hirt, J. Mol. Biol., 26:365-369). E. coli DH10B (GIBCOBRL) were transformed with the plasmid DNA so obtained. The plasmid DNAswere amplified and purified with the transformants as in (1)-3 mentionedabove. The procedures described in (1) and (2) were then repeated twice.

[0392] 2-(3) Isolation of the Positive Clone

[0393] After the third panning, transformed E. coli DH10B cells werecultivated overnight on LB plates containing ampicillin to obtaincolonies. Twenty drug-resistant colonies were cultivated, plasmid DNAswere collected by alkaline miniprep method (Maniatis et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), and the insert DNA was analyzed. Agarose gelelectrophoresis revealed that the clone having about 0.9 kb cDNA(designated “T132A7”) was concentrated.

[0394] “T132A7” was transiently expressed in COS7 cells again with themethod described in (1). After “T132A7”-introduced cells were reactedwith “JTT-1 antibody” or “JTT-2 antibody”, and then with FITC-labeledanti-mouse IgG (Cappel), the fluorescence intensity of the stained cellswas measured with EPICS-Elite flow cytometer (Coulter). “JTT-1 antibody”and “JTT-2 antibody” strongly recognized the “T132A7” gene product. Theresults are shown in FIG. 8.

[0395] 3. Determination of the Nucleotide Sequence and the Amino AcidSequence

[0396] The nucleotide sequence of clone “T132A7” was determined bydideoxy method with “Auto Read Sequencing Kit” (Pharmacia) and “A.L.F.DNA Sequencer” (Pharmacia). In addition, the deduced amino acid sequenceof “rat JTT-1 antigen” encoded by the nucleotide sequence was analyzedwith gene analysis software “GENEWORKS” (IntelliGenetics). Thenucleotide sequence and the deduced amino acid sequence were shown inSEQ ID NO: 4.

[0397] The amino acid sequence (composed of 200 amino acid residues)deduced from the cloned gene comprises the same amino acid sequence asthe N terminal amino acid sequence determined in Example 6-(3).Considering that clone “T132A7”-introduced cells strongly react with“JTT-1 antibody,” it can be concluded that clone “T132A7” comprises thecDNA encoding “rat JTT-1 antigen.”

[0398] 4. Computer Analysis

[0399] Hydropathy analysis of the primary structure of the deduced aminoacid sequence of “JTT-1 antigen” was performed according to the methodof Kite and Doolittle (Kite et al., J. Mol. Biol., 157:105-132, 1982)(FIG. 9). The results revealed that “JTT-1 antigen” is a transmembraneprotein having a signal sequence at the N-terminus. In addition, theresults of motif analysis revealed that “JTT-1 antigen” has twoAsn-linked sugar chain binding sites in the extracellular domain, andtwo casein kinase phosphorylation sites and one protein kinase Cphosphorylation site in the cytoplasmic domain. In FIG. 9 “CHO” meansN-linked sugar chain binding site; “P”, phosphorylation site; “CKII”,casein kinase II; and “PKC”, protein kinase C.

Example 8 Cloning of cDNA Encoding “Human JTT-1 Antigen”

[0400] 1. Preparation of a Probe

[0401] The cDNA (about 0.9 kb) encoding “rat JTT-1 antigen” wasgenerated by digesting the clone “T132A7” obtained in Example 7 withrestriction enzymes EcoRI and NotI, and separated by agarose gelelectrophoresis. The separated DNA fragments were purified with “QUIAEXgel extraction kit” (QUIAGEN), and the obtained DNA fragments werelabeled with ³²P using “Ready-To-Go DNA labelling kit” (Pharmacia).These labeled DNA fragments were used as probes for plaquehybridization.

[0402] 2. Preparation of cDNA Library

[0403] 2-(1) Extraction of Poly(A)⁺ RNA

[0404] Poly(A)⁺ RNA was extracted from ConA-stimulated lymphoblasts(ConA blast) derived from human peripheral blood in the same manner asin Example 7-1-(1).

[0405] 2-(2) Preparation of cDNA

[0406] With 5 μg of the poly(A)⁺ RNA so prepared as a template, cDNAswere synthesized with “oligo dT primer” (Pharmacia) and “Time Saver cDNASynthesis Kit” (Pharmacia). EcoRI adapter was then added, and sizefractionation was performed with Spun Column (Pharmacia).

[0407] 2-(3) Insertion into a Vector and Packaging

[0408] The cDNAs so obtained having EcoRI-ends were ligated with thevector “λZAPII” (Stratagene) digested with EcoRI. “DNA Ligation Kit”(Takara Shuzo) was used for the ligation. After in vitro packaging ofthe ligated DNA was performed with “GIGA PACK II GOLD” (Stratagene), E.coli XL1Blue MRF′ cells (Stratagene) were transfected with the obtainedphage particle to generate a cDNA library composed of plaque comprisingrecombinant phage.

[0409] 3. Screening of cDNA Library

[0410] cDNA library was screened by plaque hybridization method(Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.) with “Rapid hybridizationbuffer” (Amersham). First, the cDNA library so obtained (1×10⁴) wasplated onto agar plates and the replica was produced with “Hybond-Nnylon membrane” (Amersham). Plaque hybridization was performed in “Rapidhybridization buffer” (Amersham) using the replica and the ³²P-labeledprobe prepared in Example 8-1. First and second screenings wereperformed to obtain eight positive clones. Single plaques of each clonewere isolated and subjected to in vivo excision in accordance with themanual (Stratagene) and seven positive clones were collected as plasmidDNA.

[0411] 4. Determination of the Nucleotide Sequence

[0412] The nucleotide sequences of the seven clones were determined bydideoxy method with “Auto Read Sequencing Kit” (Pharmacia) and “A.L.F.DNA Sequencer” (Pharmacia). The seven clones comprise the samenucleotide sequence. It was found that clone “pBSh41” encodes the fulllength “human JTT-1 antigen.” The cDNA sequence corresponding to theopen reading frame (ORF) of “human JTT-1 antigen” is shown in SEQ ID NO:1, the full length of the deduced amino acid sequence of “human JTT-1antigen” is shown in SEQ ID NO: 2, and the nucleotide sequencecomprising 5′ and 3′ sequences is shown in SEQ ID NO: 3 (ORF correspondsto the nucleotide residues 26 to 625). It is understood that thenucleotide sequence contained in the clone encodes the full length of“human JTT-1 antigen” because the amino acid sequence (composed of 199amino acid residues) deduced from the nucleotide sequence showssignificant homology with the amino acid sequence of “rat JTT-1 antigen”(FIG. 10). As shown in FIG. 10, the homology between the amino acidsequences of human and rat “JTT-1 antigen” is 60% or more.

[0413]E. coli DH10B (GIBCO BRL) transformed with the clone “pBSh41” hasbeen deposited under the Budapest Treaty with international depositoryauthority, National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, Ministry of International Tradeand Industry, Japan (1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) sinceOct. 25, 1996 (an international deposit accession No. FERM BP-5725).

[0414] 5. Structural Characteristics and Biological Function of “JTT-1Antigen”

[0415] The results of motif search for the deduced amino acid sequenceof “human JTT-1 antigen” in known human proteins revealed that “humanJTT-1 antigen” has structural similarity to “CD28” and “CTLA-4,”human-derived cell membrane proteins belonging to the immunoglobulinsuperfamily, mentioned in detail above (FIGS. 11 and 12). As mentionedabove, “CD28” and “CTLA-4” are extremely important molecules regulatingthe activation and inhibition of T cells in immune system.

[0416] The structural similarity is as follows

[0417] 1. 20 or more amino acid residues including cysteine residues arehighly conserved.

[0418] 2. Proline repeating sequence, “Pro-Pro-Pro (PPP)”, which isessential as the ligand binding region in CD28 and CTLA-4, is conserved.

[0419] 3. “Tyr-Xaa-Xaa-Met (YxxM)” (Xaa and x represents any amino acid)sequence essential as the signal transmitting region in CD28 and CTLA-4is conserved in the cytoplasmic region.

[0420] From the fact that the same structure with the specific structureof “CD28” and “CTLA-4”, which play an important role in regulation ofactivation of T cells that are main actor in immune mechanism, “JTT-1antigen” of the present invention is inferred to play an important rolelike those molecules in regulation of activation of lymphocytes such asT cells which are main actor in immune response.

Example 9 Cloning of cDNA Encoding “Mouse JTT-1 Antigen”

[0421] 1. Preparation of a Probe

[0422] The cDNA (about 0.9 kb) encoding “rat JTT-1 antigen” was obtainedby digesting the clone “T132A7,” cloned in Example 7, with restrictionenzymes EcoRI and NotI, and separated by agarose gel electrophoresis.The DNA fragments so separated were purified with “QUIAEX gel extractionkit” (QUIAGEN), and the DNA fragments were labeled with ³²P using“Ready-To-Go DNA labelling kit” (Pharmacia). These labeled DNA fragmentswere used as a probe for plaque hybridization.

[0423] 2. Preparation of cDNA Library

[0424] 2-(1) Extraction of Poly(A)⁺ RNA

[0425] As Example 7-1-(1), poly(A)⁺ RNAs were extracted fromConA-stimulated lymphoblasts derived from mouse spleen (about 1×10⁶cells/ml).

[0426] 2-(2) Preparation of cDNA Library

[0427] With 5 mg of poly(A)⁺ RNAs prepared in the above as a template,cDNAs were synthesized with oligo dT primer (Pharmacia) and “Time SavercDNA Synthesis Kit” (Pharmacia). After EcoRI adapter was added to thecDNA, size fractionation was performed with Spun Column (Pharmacia).

[0428] 2-(3) Insertion of cDNA into a Vector and Packaging

[0429] The cDNA so obtained having EcoRI-ends was ligated with thevector 1ZAPII (Stratagene) digested with EcoRI. “DNA Ligation Kit”(Takara Shuzo) was used for the ligation. After in vitro packaging ofthe ligated DNA was performed with GIGA PACK II GOLD (Stratagene), E.coli XL1Blue MRF′ cells (Stratagene) were transfected with the phageparticle so obtained to generate a cDNA library composed of plaquecomprising recombinant phage.

[0430] 3. Screening of cDNA Library

[0431] Screening was performed by plaque hybridization method (Maniatiset al., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) using Rapid hybridization buffer(Amersham).

[0432] The above-obtained cDNA library (1×1⁰⁴) was plated onto agarplates and the replica was produced using Hybond-N nylon membrane(Amersham). Plaque hybridization was performed in Rapid hybridizationbuffer (Amersham) using the replica and the ³²P-labeled probe preparedin Example 9-1. First and second screenings were performed to obtainfive positive clones. After single plaque of each clone was isolated, invivo excision was performed in accordance with Instruction Manual(Stratagene) and five positive clones were collected as plasmid DNA.

[0433] 4. Determination of the Nucleotide Sequence

[0434] The nucleotide sequences of each of the five clones weredetermined by dideoxy method with “Auto Read Sequencing Kit” (Pharmacia)and “A.L.F. DNA Sequencer” (Pharmacia). The four of the five clonescomprise the same nucleotide sequence. The nucleotide sequence of cDNAencoding the full length of “mouse JTT-1 antigen” and the deduced aminoacid sequence are shown in SEQ ID NO: 5.

[0435] As understood from FIG. 10, “mouse JTT-1 antigen” is composed of200 amino acid residues like “rat JTT-1 antigen.” The homology among theamino acid sequences of mouse, rat, and human “JTT-1 antigens” issignificant (60% or more).

[0436] 5. Analysis of the Locus of “Mouse JTT-1 Antigen” Gene

[0437] The locus of the gene encoding “mouse JTT-1 antigen” was analyzedby fluorescence in situ hybridization method.

[0438] The cDNA so obtained encoding “mouse JTT-1 antigen” was labeledwith ³²P to prepare hybridization probes by the usual method. Usingthese probes, the 129 SVJ mouse genomic DNA library (Stratagene) wasscreened to obtain mouse genomic DNA clones comprising the exonsencoding “mouse JTT-1 antigen.” The structure of the genomic DNA isschematically shown in FIG. 13.

[0439] The above-obtained genomic DNA clones were labeled withdigoxigenin dUTP by nick translation to prepare probes. The labeledprobes were bound to cleaved mouse DNA and hybridized with normalmetaphase chromosomes derived from mouse embryonic fibroblasts in thesolution containing 50% formaldehyde, 10% dextran sulfate, and 2×SSC.After a slide glass for hybridization was incubated influorescence-labeled anti-digoxigenin antibody, specific hybridizationsignal was detected by staining with DAPI. In the first test, the partnear the largest chromosome that was thought to be the chromosome 1,judging from the DNA size and emerged band, was specifically labeled.Based on this information, the above-described genomic DNA clone wasco-hybridized with probes specific to the centromere region of thechromosome 1. As a result, the centromere region of the chromosome 1 andthe regions near them were specifically labeled. Ten samples of thechromosome 1 showing the specific hybridization were analyzed, and itwas revealed that the above-mentioned genomic DNA clone was located atthe position of 33% of the distance from the border betweenheterochromatin and euchromatin to the telomere of the chromosome 1,namely, on the same band “1C3” as the loci of mouse “CD28” and “CTLA-4”genes. As the result that 80 metaphase cells were analyzed, specificlabeling was identified at said position for 79 cells.

[0440] These results and the results obtained in Example 8 indicatingthe structural similarity of “JTT-1 antigen” to “CD28” and “CTLA-4”suggest that “JTT-1 antigen,” like “CD28” and “CTLA-4,” is an importantmolecule involved in the regulation of the transmission of costimulatorysignal and/or activation of lymphocytes.

Example 10 Cloning of cDNA Encoding a Mutant of “Rat JTT-1 Antigen”

[0441] Another cDNA that is thought to encode alternative splicingvariant of “rat JTT-1 antigen” cloned in Example 7 was cloned asfollows.

[0442] 1. Preparation of a Probe

[0443] The cDNA (about 0.9 kb) encoding “rat JTT-1 antigen” wasgenerated by digesting the clone “T132A7,” obtained in Example 7, withrestriction enzymes EcoRI and NotI, and separated by agarose gelelectrophoresis. The separated DNA fragments were purified with “QUIAEXgel extraction kit” (QUIAGEN), and the obtained DNA fragments werelabeled with ³²P using “Ready-To-Go DNA labeling kit” (Pharmacia). Theselabeled DNA fragments were used as probes for plaque hybridization.

[0444] 2. Preparation of cDNA Library

[0445] 2-(1) Extraction of Poly(A)⁺ RNA

[0446] As in Example 7-1-(1), poly(A)⁺ RNA was extracted from ratthymoma cell line FTL435 (about 1×10⁶ cells/ml)

[0447] 2-(2) Preparation of cDNA Library

[0448] With 5 mg of the poly(A)⁺ RNA prepared as mentioned above as atemplate, cDNAs were synthesized using oligo dT primer (Pharmacia) and“Time Saver cDNA Synthesis Kit” (Pharmacia). After EcoRI adapter wasadded to the cDNA, size fractionation was performed with Spun Column(Pharmacia).

[0449] 2-(3) Insertion of cDNA into a Vector and Packaging

[0450] The cDNA having EcoRI-end obtained above was ligated with thevector 1ZAPII (Stratagene) digested with EcoRI. “DNA Ligation Kit”(Takara Shuzo) was used for ligation. After in vitro packaging of theligated DNA was performed with GIGA PACK II GOLD (Stratagene), E. coliXL1Blue MRF′ (Stratagene) was transfected with the obtained phageparticle to generate a cDNA library composed of plaque comprisingrecombinant phage.

[0451] 3. Screening of cDNA Library

[0452] Screening was performed by plaque hybridization method (Maniatiset al., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) with Rapid hybridization buffer(Amersham).

[0453] The above-prepared cDNA library (1×10⁴) was plated onto agarplates and the replica was produced with Hybond-N nylon membrane(Amersham). Plaque hybridization was performed in Rapid hybridizationbuffer (Amersham) using the replica and the ³²P-labeled probe preparedin Example 10-1. First and second screenings were performed to obtaintwo positive clones. After single plaque of each clone was isolated, invivo excision was performed in accordance with Instruction Manual(Stratagene), and two positive clones were collected as plasmid DNA.

[0454] 4. Determination of the Nucleotide Sequence

[0455] The nucleotide sequences of the two clones were determined bydideoxy method with “Auto Read Sequencing Kit” (Pharmacia) and A.L.F.DNA Sequencer (Pharmacia). The two clones comprise the same nucleotidesequence. The nucleotide sequence of cDNA encoding the full length ofthe obtained “rat JTT-1 antigen” and the deduced amino acid sequence areshown in SEQ ID NO: 6. The amino acid sequence (SEQ ID NO: 6) deducedfrom the obtained cDNA sequence was compared with the amino acidsequence (SEQ ID NO: 4) deduced from the obtained cDNA sequence encoding“rat JTT-1 antigen” cloned in Example 7 (FIG. 14). As shown in FIG. 14,the amino acid sequence encoded by the cDNA cloned in this test wascompletely the same as that encoded by the cDNA encoding “rat JTT-1antigen” obtained in Example 7, except that (1) C-terminal threecontinuous amino acid residues (Met-ThrSer) changes into Thr-Ala-Pro,and that (2) subsequent to the Thr-Ala-Pro, 16 continuous amino acidresidues(Leu-ArgAla-Leu-Gly-Arg-Gly-Glu-His-Ser-Ser-Cys-Gln-Asp-Arg-Asn) areadded. This indicates that the cDNA cloned in this test encodes thealternative splicing variant of “rat JTT-1 antigen” obtained in Example7.

Example 11 Preparation of Recombinant “Human JTT-1 Antigen”-ExpressingCells

[0456] The plasmid clone pBSh41 obtained in Example 8 was digested witha restriction enzyme EcoRI, and a DNA fragment comprising the cDNAencoding the full length of “human JTT-1 antigen” was excised. This DNAfragment was inserted with DNA Ligation Kit (Takara Shuzo) into aplasmid pEFneo (Proc. Natl. Acad. Sci. USA 91:158-162, 1994) treatedwith the same restriction enzyme EcoRI to prepare the expression vector.CHO-K1 cells (ATCC: CCL-61) were transformed with the vector byelectroporation. By cultivating the cells in RPMI1640 medium containing0.8 mg/ml Geneticin (GIBCO BRL) and 10% fetal calf serum for about twoweeks, Geneticin-resistant transformants were selected. The expressionof recombinant “human JTT-1 antigen” was confirmed by Northern blottingby the usual method.

Example 12 Preparation of Monoclonal Antibodies Against “Human JTT-1Antigen”

[0457] The recombinant “human JTT-1 antigen”-expressing transformantsprepared in Example 11 were homogenized and ultracentrifuged(100,000×g). The pellet containing the cell membrane fraction wascollected and suspended in PBS. The resulting suspension comprising thecell membrane fraction was injected into the footpad of a BALB/c mousewith complete Freund's adjuvant for the first immunization (day 0). Thecell membrane fraction antigen was further administered into its footpadat intervals of 7, 14, and 28 days. Two days after the lastimmunization, the lymph node cells was taken out. The lymph node cellsand mouse myeloma cells PAI (JCR No. B0113; Res. Disclosure 217:155,1982) were mixed at a ratio of 5:1, and fused using polyethyleneglycol4000 (GIBCO) as a fusing agent to prepare monoclonal antibody-producinghybridomas. The hybridomas were screened by cultivating them inHAT-containing ASF104 medium (Ajinomoto) supplemented with 10% fetalcalf serum and aminopterin. The culture supernatant of each hybridomawas reacted with the recombinant “human JTT-1 antigen”-expressingtransformants prepared in Example 11, and the fluorescence intensity ofcells stained by reacting them with FITC-labeled anti-mouse IgG (Cappel)was measured with EPICS-ELITE flow cytometer to confirm the reactivityof the monoclonal antibody generated in each culture supernatant to“human JTT-1 antigen.” It has been confirmed that 10 or more kinds ofhybridomas producing monoclonal antibodies reactive to “human JTT-1antigen” were obtained.

[0458] Each of two kinds (designated clone SA12 and SG430) among thesehybridomas (10⁶ to 10⁷ cells/0.5 ml/mouse) was injected into a ICR nu/numouse (female, 7-8 weeks old) intraperitoneally. After 10 to 20 days,celiotomy of the mice was performed under anesthesia, and the two kindsof monoclonal antibodies (SA12 and SG430) reactive to “human JTT-1antigen” were prepared in a large amount from the ascites fluidextracted by the usual method.

Example 13 Effect of the Monoclonal Antibodies Against “Human JTT-1Antigen” on Human Peripheral Blood Lymphocytes

[0459] As mentioned in Example 8, it is thought that “JTT-1 antigen” canbe involved in the regulation of the activation of lymphocytes in immunereaction like “CD28” and “CTLA-4.” In order to prove this, the effect ofthe monoclonal antibodies against “human JTT-1 antigen” on humanlymphocytes was analyzed in light of cell growth as an indication.

[0460] To each well of 96-well microtiter plate were added (1) eitherSA12 or SG430 (1 μg/ml), the monoclonal antibody against “human JTT-1antigen” prepared in Example 12, or (2) a mixture of either monoclonalantibody SA12 or SG430 (1 μg/ml) with anti-CD3 monoclonal antibody OKT-3(1 μg/ml, Orthodiagnostic Systems), which is used for adding the primarysignal in the activation of lymphocytes. The plate was incubated at 37°C. for 1 hour to coat each well with the antibody. After the plate waswashed with RPMI1640 medium, normal human peripheral blood lymphocytes(1×10⁵ cells/well) were added to each well and incubated in RPMI1640medium containing 10% fetal calf serum for 3 days. If necessary, 1 ng/mlphorbol myristate acetate (PMA) was added. Then, [³H] thymidine (3.7μkBq/well) was added to each well, and the plate was incubated at 37° C.for 6 hours. The cells were harvested, and the amount of [³H] thymidineincorporated into DNA was measured with a liquid scintillation counter(Beckman). The assay without any antibody was used as a control. Theresults are shown in FIG. 15.

[0461] In the assay using the plates coated with either monoclonalantibody SA12 or SG430, the number of lymphocytes increased about 10times compared to the control. In the co-presence of OKT3, the number oflymphocytes increased about 100 times when either monoclonal antibodySA12 or SG430 was used.

[0462] These results indicate that “JTT-1 antigen” functions in theregulation of the lymphocyte activation. The fact that the cell growthrate was increased by using together with OKT3 indicates that “JTT-1antigen” is involved in the transmission of costimulatory signal like“CD28” and “CTLA-4.”

Example 14 Effect of “JTT-2 Antibody” on Experimental AllergicEncephalomyelitis (EAE)

[0463] As above mentioned in detail, recently, many attempts to treatvarious autoimmune diseases (rheumatoid arthritis, multiple sclerosis,autoimmune thyroiditis, allergic contact dermatitis, chronicinflammatory dermatosis such as lichen planus, systemic lupuserythematosus, insulin dependent diabetes mellitus, psoriasis, etc.)have been made by regulating the transmission of between CD28/CTLA-4 andCD80/CD86. The effect has been already confirmed in various modelanimals of autoimmune diseases ((1) a model for human systemic lupuserythematosus (SLE); (2) experimental allergic encephalomyelitis (EAE),a model for multiple sclerosis (MS); (3) a model for insulin dependentdiabetes mellitus (IDDM); (4) Goodpasture's nephritis model; and (5)human rheumatoid arthritis).

[0464] In order to determine whether “JTT-1 antigen” of the presentinvention is a molecule involved in the activation or inhibition oflymphocytes such as “CD28” and “CTLA-4,” model rats for experimentalallergic encephalomyelitis (EAE), a model for multiple sclerosis (MS),were produced, and the effect of the titled monoclonal antibody on“JTT-1 antigen” in the model was analyzed.

[0465] An emulsion to be used as immunogen was prepared by mixingHartley guinea pig cerebrospinal homogenate (800 mg/ml physiologicalsaline) with the same amount of Freund's complete adjuvant. Immunizationwas performed by intradermally injecting the emulsion into left andright foot pads of 15 Lewis rats (female, 6-week-old) in an amount of0.25 ml per footpad. The administration (immunization) was adjusted soas for the dosages of the homogenate prepared to be 200 mg per rat. Thisimmunization so induces experimental allergic encephalomyelitis (EAE).

[0466] The rats so immunized were divided into three groups of five ratseach, and any one of (1) to (3) below was intravenously injected intomice of each group immediately after immunization (day 0), and 3, 6, 9,and 12 days after the immunization.

[0467] (1) Monoclonal antibody “JTT-2 antibody” against “rat JTT-1antigen” prepared in Example 2 (dosage: 2 mg/ml PBS, 5 mg/kg)

[0468] (2) Prednisolone, steroid agent (dosage: 4 mg/ml PBS, 10 mg/kg)

[0469] (3) Control antibody non-reactive to “rat JTT-1 antigen” (dosage:2 mg/ml PBS, 5 mg/kg)

[0470] Symptom was observed in the course of time after theimmunization. After the onset of EAE had been found, the degree of thesymptom was estimated by scoring the symptom based on the followingcriteria.

[0471] (Score 1) Disappearance of tension of a tail

[0472] (Score 2) Dragging of hind legs, and slight paralysis

[0473] (Score 3) Dragging of hind legs, and serious paralysis

[0474] (Score 4) Paralysis of the whole body, or death

[0475] The results are shown in FIG. 16. In the group to which thecontrol antibody was administered, the symptom of EAE reached the peak(maximum score) at day 11 to 15 after the immunization, and thengradually recovered. In contrast, in the “JTT-2 antibody”-administeredgroup, the symptom of EAE at day 11 after the immunization wassignificantly inhibited. This inhibitory effect was significantly higherthan that in the prednisolone-administered group.

[0476] These results indicate that “JTT-1 antigen” is a molecule thatfunctions in the induction of immune response such as the lymphocyteactivation induced by immunization by foreign antigens, and that theregulation of the function of “JTT-1 antigen” or its ligands can inhibitthe symptom of various autoimmune diseases.

Example 15 Effect of “JTT-2 Antibody” on Glomerulonephritis

[0477] For the same purpose as Example 14, glomerulus basement membrane(GBM) nephritis model rats were produced, and the effect of the titledmonoclonal antibody on “JTT-1 antigen” in the model was analyzed.

[0478] After bovine glomerulus basement membrane (Shigei MedicalInstitute) digested with collagenase was diluted with physiologicalsaline to 200 μg/ml, the dilution was mixed with Freund's completeadjuvant to prepare an emulsion to be used as immunogen. Immunizationwas performed by intradermally injecting the emulsion into both hindsoles of 48 Wistar kyoto rats (about 200 g) under anesthesia in anamount of about 0.2 ml per footpad (dosage: about 15 μg). Thisimmunization so induces glomerulus basement membrane (GBM) nephritis.

[0479] The immunized rats were divided into eight groups of six ratseach, and any one of (1) to (3) below was injected into rats of eachgroup immediately after immunization (day 0), and three times a week for5 consecutive weeks.

[0480] (1) Monoclonal antibody “JTT-2 antibody” against “rat JTT-1antigen” prepared in Example 2 (dosage: 3 mg/kg (2 ml PBS/kg),intravenous injection)

[0481] (2) Prednisolone, steroid agent, as a positive control (suspendedin 0.5% carboxymethylcellulose (CMC)) (dosage: 3 mg/kg (5 ml/kg), oraladministration)

[0482] (3) 0.5% CMC as a negative control (dosage: 5 ml/kg, oraladministration)

[0483] After the administration of a test substrate, sterilized water(25 ml/kg) was orally administered into each rat forcedly, and urine wascollected for 5 hours from each rat which had been kept in a metabolismcage without eating and drinking. After the volume of the collectedurine was measured, the urinary protein concentration was measured usingTonein TP-II (Otuka), and the urinary excretion of protein per fivehours was calculated (unit: mg protein/5 hours). The above-mentionedurinary collection and urinary protein measurement were performed in thesame manner at 1, 2, 3, and 4 weeks after the immunization (day 0).

[0484] The results are shown in FIG. 17. Compared to the control group,the urinary excretion of protein at 3 weeks after the immunization wassignificantly reduced in the “JTT-2 antibody”-administered group.

[0485] These results indicate that “JTT-1 antigen” is a molecule thatinduces immune response such as the lymphocyte activation induced byimmunization by foreign antigens, and that the regulation of thefunction of “JTT-1 antigen” or its ligands can inhibit the symptom ofvarious autoimmune diseases.

Example 16 Preparation of the Fusion Protein Between “JTT-1 Antigen” andIgFc

[0486] As mentioned in Examples 8, and 13 to 15, “JTT-1 antigen” of thepresent invention is thought to be a molecule such as “CD28” and“CTLA-4” involved in the transmission of costimulatory signal involvedin the regulation of the activation of lymphocytes. In addition, asmentioned in Example 14, a fusion protein (CTLA-4-IgFc) composed of theextracellular domain of “CTLA-4” and the Fc region of humanimmunoglobulin IgG1 reportedly has therapeutic effects on variousautoimmune diseases. In this Example, a fusion protein composed of theextracellular region of “JTT-1 antigen” and human IgGFc was prepared asfollows in order to examine whether soluble JTT-1 antigen, likeCTLA-4-IgFc, could be applied to therapy of various autoimmune diseases.

[0487] (1) Preparation of the Fusion Protein Between “Rat JTT-1 Antigen”and Human IgG1-Fc (rJTT-1-IgFc)

[0488] In order to amplify the cDNA encoding the extracellular region of“rat JTT-1 antigen” by PCR, 5′ primer having XhoI restriction site(5′CTGCTCGAGATGAAGCCCTACTTCTCG-3′, SEQ ID NO: 7) and 3′ primer havingBamHI restriction site (5′ACCCTACGGGTAACGGATCCTTCAGCTGGCAA-3′, SEQ IDNO:8) at their terminus were designed and synthesized. Using cDNA clone“T132A7” obtained in Example 7 encoding the full length of “rat JTT-1antigen” as a template, PCR was performed with the primers to preparethe cDNA comprising the cDNA encoding the extracellular region of “ratJTT-1 antigen” having XhoI and BamHI restriction sites at its both ends.The PCR products so obtained were digested with XhoI and BamHI andseparated by agarose gel electrophoresis to isolate an about 450-bp bandpredicted to be the cDNA fragment encoding a desired extracellularregion. The isolated cDNA fragment was subcloned into pBluescript II SK(+) (Stratagene) cleaved with XhoI and BamHI. Sequence analysis with anautomated fluorescence DNA sequencer (Applied Biosystems) revealed thatthe cDNA fragment comprises the region encoding amino acid sequencecorresponding to the amino acid residues 1 to 141 of “rat JTT-1 antigen”(SEQ ID NO: 4).

[0489] On the other hand, the DNA encoding the Fc of human IgG1 as thefusion partner was cut out as an about 1.3 kb BamHI-XbaI DNA fragment bydigesting the plasmid (see Cell 61:1303-1313, 1990). Prepared by B. Seedet al. (Massachusetts General Hospital)) with BamHI and XbaI. Thisfragment comprises exons encoding human IgG1 hinge region, Cγ₁2, andCγ₁3.

[0490] The XhoI-BamHI fragment encoding the extracellular region of “ratJTT-1 antigen,” and BamHI-XbaI fragment comprising exons encoding the Fcof human IgG1 (“IgFc”), both prepared as mentioned above, were subclonedinto pBluescript II SK (+) (Stratagene) cleaved with XhoI and XbaI.

[0491] Then, the plasmid was digested with XhoI and XbaI, and an about1.8 kb DNA fragment comprising the fusion DNA comprising theextracellular region of “rat JTT-1 antigen” and human IgFc was cut out.This fusion DNA fragment was inserted into the XhoI and XbaI sites ofthe expression vector pME18S (Medical Immunology 20:27-32, 1990;Experimental Medicine: SUPPLEMENT, “Handbook of Genetic Engineering,”Yodosha, pp. 101-107, 1992) with T4 DNA ligase to construct plasmidprJTT-1-IgFc.

[0492] HEK293 cells (ATCC CRL1573) subconfluently cultivated asmonolayer in DMEM medium containing 10% fetal calf serum and ampicillinwere transformed with prJTT-1-IgFc by electroporation to obtaintransformants.

[0493] The transformants were cultured in serum-free ASF104 medium for72 hours to express rJTT-1-IgFc.

[0494] Using a Protein G Sepharose affinity column (Pharmacia),rJTT-1-IgFc was purified as follows.

[0495] The supernatant obtained by centrifuging the culture mediummentioned above was loaded onto Protein G Sepharose affinity columnpreviously equilibrated with binding buffer. After the column was washedwith binding buffer, elution was performed with elution buffer. Theeluate was collected and dialyzed against phosphate buffer withexchanging the external solution twice or more to obtain purerJTT-1-IgFc.

[0496] The result of affinity chromatography is shown in FIG. 18, andthe result of SDS-PAGE of the pure rJTT-1-IgFc so obtained in FIG. 19.

[0497] (2) Preparation of the Fusion Protein Between “Human JTT-1Antigen” and Human IgG1-Fc (hJTT-1-IgFc)

[0498] hJTT-1-IgFc was prepared as mentioned above in (1), except forcDNA used as templates and primers for PCR. In this test, the clone“pBSh41” comprising the cDNA encoding the full length “human JTT-1antigen” prepared in Example 8 was used as a template, and5′TAACTGTTTCTCGAGAACATGAAGTCAGGC-3′ (SEQ ID NO: 9) and5′ATCCTATGGGTAACGGATCCTTCAGCTGGC-3′ (SEQ ID NO: 10) were used asprimers.

[0499] The result of affinity chromatography is shown in FIG. 20, andthe result of SDS-PAGE of the pure hJTT-1-IgFc so obtained in FIG. 21.

Example 17 Preparation of a Transgenic Mouse in Which cDNA Encoding “ratJTT-1 Antigen” has Been Integrated

[0500] The cDNA encoding the full length of “rat JTT-1 antigen” obtainedin Example 7 was inserted into the expression vector pCAGGS (Gene108:193-200, 1991) having chicken β actin promoter using DNA BluntingKit (Takara) to obtain plasmid, prJTT-1. In order to prepare atransgenic mouse, prJTT-1 was linearized by restriction enzymetreatment.

[0501] A female ICR mouse having a vaginal plug, obtained by mating awhite ICR mouse (Nihon LSC) with a male vasoligated white ICR mouse(Nihon SLC), was used as a foster mother mouse. A mouse for obtainingfertilized eggs for introducing “rat JTT-1 antigen” gene thereinto wasprepared by mating a female BDF-1 mouse (Nihon SLC) that had been madeto superovulate by administered PEAMEX (5 units, Sankyo Zoki) andPregnil (5 units, Organon) with a male BDF1 male (Nihon SLC). Aftermating, the oviduct was excised from the female BDF-1 mouse, and onlyfertilized eggs were obtained by hyaluronidase treatment and stored in amedium.

[0502] The “rat JTT-1 antigen” gene was introduced into the fertilizedegg under microscopy using a manipulator according to the usual method.The fertilized egg was fixed with a retaining needle. A solutioncontaining the above-mentioned linearized gene encoding “rat JTT-1antigen,” which was diluted with Tris-EDTA buffer, was microinjectedinto the male pronucleus of the fertilized eggs with a DNA introductionneedle at 37° C.

[0503] After gene introduction, only fertilized eggs keeping normalstate were selected, and then, the fertilized egg so selected in whichthe “rat JTT-1 antigen” genes have been introduced was inserted into theovarian fimbria in the ovary of a foster mother mouse (white ICR mouse).

[0504] The tail of a progeny mouse (founder mouse) born from the fostermother mouse was cut off and the genomic gene was collected from it. Itwas confirmed by PCR that the “rat JTT-1 antigen” gene was integratedinto the mouse genome. Then, heterozygous transgenic mice highlyexpressing “rat JTT-1 antigen” were prepared by mating this foundermouse with a normal mouse. Homozygous transgenic mice can be prepared bymating the heterozygous mice with each other.

[0505] The microinjected construct comprising the “rat JTT1 antigen”gene is schematically shown in FIG. 22.

Example 18 Preparation of a Knockout Mouse Whose Endogenous GeneEncoding “Mouse JTT-1 Antigen” has Been Inactivated

[0506] (1) Construction of a Targeting Vector

[0507] A targeting vector for inactivating (knocking out) the endogenousgene encoding “mouse JTT-1 antigen” through homologous recombination(Nikkei Science, pp. 52-62, May 1994) was prepared as follows.

[0508] The PstI-HindIII fragment (“homologous DNA (1)”) obtained bydigesting the mouse genomic DNA clone comprising the region encoding“mouse JTT-1 antigen” cloned in Example 9-5 with PstI and HindIII wassubcloned into pGEM-3 (Promega). Then, pGEM-3 was linearized with XhoI,and neomycin resistance gene (“neo”) excised from pMC1-neo-polyA(Stratagene) by treating it with XhoI and SalI was inserted at theupstream of the “homologous DNA” (1) and then ligated them. Theabove-mentioned mouse genomic DNA clone was digested with XhoI and NotIto cut off an about 5.5 kb gene (“homologous DNA (2)”) located upstreamof above-mentioned “homologous DNA (1).” Separately, the above-mentionedpGEM3 into which “neo-homologous DNA (1)” has been inserted was digestedwith XhoI and HindIII to cut off “neo-homologous DNA (1).” “HomologousDNA (2)” and “neo-homologous DNA (1)” thus obtained were subcloned intopSEAP2-CONT (Clontech) linearized with NotI and HindIII.

[0509] After the obtained plasmid, in which “homologous(2)-neo-homologous (1)” has been inserted, was digested and linearizedat the downstream of “homologous DNA (1) with NruI, thymidine kinasegene (“TK”) obtained by digesting pMC1-TK (Stratagene) with PvuII wasinserted at the downstream of “homologous DNA (1)” to obtain a targetingvector, in which “homologous DNA (2)-neo-homologous (1)” was inserted.

[0510] (2) Introduction of the Targeting Vector into ES Cells

[0511] Mouse embryonic stem cells (Nature 362:255-258, 1993; Nature326:292-295, 1987) cultured in DMEM medium containing 15% fetal calfserum were treated with trypsin to be single cells, and the cells werewashed three times, followed by adding phosphate buffer thereto toadjust 1×10⁷ cells/ml. The targeting vector mentioned above (25 μg per 1ml of the cell suspension) was added to the cell suspension, andelectric pulse was delivered once under the condition of 350 V/cm (25μF). Then, 1×10⁷ of ES cells were plated on a 10-cm dish and cultivatedin maintenance medium for a day, and the medium was changed to selectionmedium (containing 250 μg/ml G418 and 2 μM ganciclovir). The cells werecultivated with the medium changed every two days. At the tenth day fromthe introduction of the targeting vector, 573 neomycin-resistant ES cellclones were obtained under microscopy with a micropipet. Each of the EScell clones so obtained were cultivated independently on a 24-well platecoated by Feeder cells to obtain 768 neomycin-resistant ES cellreplicas.

[0512] (3) Screening of Knockout ES Cells

[0513] It was confirmed by PCR whether the endogenous gene encoding“mouse JTT-1 antigen” was disrupted (knocked out) through homologousrecombination in each of the neomycin-resistant ES cells.

[0514] For PCR, (1) primers designed and synthesized based on thesequence of above-mentioned neomycin-resistant gene (“neo”)(5′-CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC-3′, SEQ ID NO: 11) and (2)primers designed and synthesized based on the sequence ofabove-mentioned “homologous DNA (1)”(5′CATTCAAGTTTCAGGGAACTAGTCCATGCGTTTC-3′, SEQ ID NO: 12) were used.

[0515] Each genomic DNA was extracted from each of theneomycin-resistant ES cell, and PCRs were performed using the primerswith the genomic DNA as a template. PCR was performed 1 cycle ofreaction at 94° C. for 3 minutes, 30 cycles of reaction at 94° C. for 1minute, at 60° C. for 1 minute, and at 72° C. for 3.5 minutes, and 1cycle of reaction at 72° C. for 10 minutes, and the resulting productswere stored at 4° C. When a fragment less than about 4 kb was amplifiedby this PCR, it could be judged that the endogenous gene encoding “mouseJTT-1 antigen” was disrupted (knocked out) through homologousrecombination in the ES cell clone.

[0516] A desired PCR product was obtained from three of 768 ES cellclones tested. Genomic southern blottings were performed for these threeclones for further screening and confirmation. After genomic DNA wasextracted from the three clones and digested with restriction enzymeBamHI, the digested products were subjected to agarose gelelectrophoresis. The resulting DNAs were transferred to nylon membrane,and hybridization was performed with a probe prepared from the genomicDNA sequence comprising “mouse JTT-1.” The probe was designed based onthe sequence outside the site where homologous recombination occurred,which enables to distinguish mutant type genome from normal type genomein size.

[0517] As a result, two bands corresponding to mutant type and normaltype were observed in one of the three clones. This ES cell clone wasused for the preparation of a knockout mouse described below.

[0518] (4) Preparation of a Knockout Mouse

[0519] The above-obtained ES cells (15 ES cells per blastocyst) whoseendogenous gene encoding “mouse JTT-1 antigen” has been inactivated(knocked out) through homologous recombination were microinjected intoblastocysts to, which were obtained by mating a female C57BL6 mouse(Nihon Charles River) with male one. Immediately after themicroinjection, the blastocysts (about 10 blastocysts per one side ofthe uterus) were transplanted in the uterus of a foster mother ICR mouse(CLEA Japan), which was 2.5 day-mouse from pseudopregnant treatment. Asa result, 38 progeny mice in total were obtained, and 18 out of themwere desired chimeric mice. Eleven (11) out of the chimeric mice werethe chimeric-mouse in which the contribution to hair color was 80% ormore.

[0520] The chimeric mice so obtained were then mated with a normalC57BL6 mice to obtain agouti mice whose color is derived from hair colorgene of the ES cells.

Example 19 Preparation of Pharmaceutical Composition Comprising Antibody

[0521] Each of the monoclonal antibody (50-150 μg/ml), “JTT-1 antibody”and “JTT-2 antibody” against “rat JTT-1 antigen,” prepared in Example 1,and monoclonal antibodies, “SA12” and “SG430” against “human JTT-1antigen,” prepared in Example 12, was added to injectable distilledwater (10 ml) to prepare injection.

INDUSTRIAL APPLICABILITY

[0522] Novel cell surface molecules (called “JTT-1 antigen”) of thepresent invention derived from mammals such as human, mouse, and rat arecharacterized as follows.

[0523] (1) “JTT-1 antigen” had the following similarity with “CD28,” acell surface molecule on lymphocytes such as T cells, which transmitscostimulatory signal important for T cell activation through celladhesion, and “CTLA-4,” a cell surface molecule on lymphocytes such as Tcells, which regulates the function of activated lymphocytes such asactivated T cells, cooperating with the signal.

[0524] (i) 20 or more amino acid residues including cysteine residuesare highly conserved;

[0525] (ii) Proline repeating sequence, “Pro-Pro-Pro (PPP),” which isessential as the ligand binding region, is conserved in theextracellular region;

[0526] (iii) “Tyr-Xaa-Xaa-Met (YxxM)” (Xaa and x represents any aminoacid) sequence essential as the signal transmitting region is conservedin the cytoplasmic region; and

[0527] (iv) The locus of the gene encoding “mouse JTT1 antigen” on mousechromosome is “1C3”, like “CD28” and “CTLA-4.”

[0528] (2) “JTT-1 antigen” can mediate cell adhesion of thymocytes,lymphoblasts stimulated with mitogen such as ConA, thymomas, like “CD28”and “CTLA-4” that mediate cell adhesion.

[0529] (3) “JTT-1 antigen” is strongly expressed, at least, inthymocytes, lymphoblast cells stimulated with mitogen such as ConA(activated T lymphoblast cells and activated B lymphoblast cells, etc.),peripheral blood lymphocytes, and thymomas.

[0530] (4) The antibody against “JTT-1 antigen” significantlyproliferates human peripheral blood lymphocytes, and the proliferationis more enhanced in the presence of a monoclonal antibody against CD3constituting TcR/CD3 complex on T cells that receive the primary signalessential for T cell activation from antigen-presenting cells.

[0531] (5) The administration of the antibody against “JTT-1 antigen”significantly inhibits the symptom of experimental allergicencephalomyelitis (EAE).

[0532] (6) The administration of the antibody against “JTT-1 antigen” toa model rat for glomerulus basement membrane (GBM) nephritissignificantly inhibits the symptom of this disease.

[0533] “JTT-1 antigen” of the present invention is, like “CD28” and“CTLA-4,” thought to be a molecule transmitting the secondary signal(costimulatory signal) essential for the activation of lymphocytes suchas T cells, and regulating the function of activated lymphocytes such asactivated T cells, cooperating with the signal.

[0534] Therefore, polypeptides constituting such cell surface molecules,its polypeptide fragment, and fusion polypeptides therefrom, andantibodies thereto of the present invention can provide extremely usefulpharmaceuticals for therapy or prevention of various autoimmunediseases, allergic diseases, or inflammatory diseases, specifically,rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis,allergic contact dermatitis, chronic inflammatory dermatosis such aslichen planus, systemic lupus erythematosus, insulin dependent diabetesmellitus, and psoriasis, caused by the activation of lymphocytes such asT cells and the abnormality of regulation of activated lymphocytefunctions.

[0535] Similarly, the genes encoding polypeptides or polypeptidefragments of the present invention can be used in not only gene therapyof various diseases as mentioned above but also preparation of antisensepharmaceuticals.

[0536] Among the antibodies of the present invention, human monoclonalantibodies and their pharmaceutical compositions have dramaticallyincreased pharmaceutical value of antibody drugs because they have noantigenicity against human, which has been a serious problem (sideeffect) of antibody pharmaceuticals containing nonhuman mammal-derivedantibodies such as mouse-derived antibodies.

[0537] The genes (DNA), polypeptides, polypeptide fragments andantibodies of the present invention are useful not only aspharmaceuticals but also as reagents for searching molecules (ligands)interacting with the cell surface molecules of the present invention,clarifying the function of the ligand, and developing drugs targetingthe ligands.

[0538] Furthermore, the transgenic mouse of the present invention isextremely useful not only as a model animal for studying physiologicalfunction of “JTT-1 antigen” that is a cell surface molecule of thepresent invention but also as a tool for screening various drugs (lowmolecular weight compounds, antibodies, antisense substances,polypeptides, etc.) having activity regulating (inhibition, suppression,activation, stimulation, etc.) the function of “JTT-1 antigen.”Specifically, such test substances can be administered to the transgenicmouse to measure and analyze various physiological, biological, orpharmacological parameters generated in the mouse, thereby assessingactivity of the administered test substances.

[0539] In addition, the knockout mouse of the present invention canclarify the function of the cell surface molecules of the presentinvention by analyzing the characteristics of the mouse from variousviewpoints (physiological, biological, pharmacological, pathological,and genetic viewpoints).

1 26 1 600 DNA Homo sapiens CDS (1)...(597) 1 atg aag tca ggc ctc tggtat ttc ttt ctc ttc tgc ttg cgc att aaa 48 Met Lys Ser Gly Leu Trp TyrPhe Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 gtt tta aca gga gaa atcaat ggt tct gcc aat tat gag atg ttt ata 96 Val Leu Thr Gly Glu Ile AsnGly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 ttt cac aac gga ggt gta caaatt tta tgc aaa tat cct gac att gtc 144 Phe His Asn Gly Gly Val Gln IleLeu Cys Lys Tyr Pro Asp Ile Val 35 40 45 cag caa ttt aaa atg cag ttg ctgaaa ggg ggg caa ata ctc tgc gat 192 Gln Gln Phe Lys Met Gln Leu Leu LysGly Gly Gln Ile Leu Cys Asp 50 55 60 ctc act aag aca aaa gga agt gga aacaca gtg tcc att aag agt ctg 240 Leu Thr Lys Thr Lys Gly Ser Gly Asn ThrVal Ser Ile Lys Ser Leu 65 70 75 80 aaa ttc tgc cat tct cag tta tcc aacaac agt gtc tct ttt ttt cta 288 Lys Phe Cys His Ser Gln Leu Ser Asn AsnSer Val Ser Phe Phe Leu 85 90 95 tac aac ttg gac cat tct cat gcc aac tattac ttc tgc aac cta tca 336 Tyr Asn Leu Asp His Ser His Ala Asn Tyr TyrPhe Cys Asn Leu Ser 100 105 110 att ttt gat cct cct cct ttt aaa gta actctt aca gga gga tat ttg 384 Ile Phe Asp Pro Pro Pro Phe Lys Val Thr LeuThr Gly Gly Tyr Leu 115 120 125 cat att tat gaa tca caa ctt tgt tgc cagctg aag ttc tgg tta ccc 432 His Ile Tyr Glu Ser Gln Leu Cys Cys Gln LeuLys Phe Trp Leu Pro 130 135 140 ata gga tgt gca gcc ttt gtt gta gtc tgcatt ttg gga tgc ata ctt 480 Ile Gly Cys Ala Ala Phe Val Val Val Cys IleLeu Gly Cys Ile Leu 145 150 155 160 att tgt tgg ctt aca aaa aag aag tattca tcc agt gtg cac gac cct 528 Ile Cys Trp Leu Thr Lys Lys Lys Tyr SerSer Ser Val His Asp Pro 165 170 175 aac ggt gaa tac atg ttc atg aga gcagtg aac aca gcc aaa aaa tct 576 Asn Gly Glu Tyr Met Phe Met Arg Ala ValAsn Thr Ala Lys Lys Ser 180 185 190 aga ctc aca gat gtg acc cta taa 600Arg Leu Thr Asp Val Thr Leu 195 2 199 PRT Homo sapiens 2 Met Lys Ser GlyLeu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 Val Leu ThrGly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 Phe His AsnGly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45 Gln Gln PheLys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50 55 60 Leu Thr LysThr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu 65 70 75 80 Lys PheCys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95 Tyr AsnLeu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser 100 105 110 IlePhe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu 115 120 125His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro 130 135140 Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu 145150 155 160 Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His AspPro 165 170 175 Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala LysLys Ser 180 185 190 Arg Leu Thr Asp Val Thr Leu 195 3 2610 DNA Homosapiens CDS (26)...(622) 3 ggactgttaa ctgtttctgg caaac atg aag tca ggcctc tgg tat ttc ttt 52 Met Lys Ser Gly Leu Trp Tyr Phe Phe 1 5 ctc ttctgc ttg cgc att aaa gtt tta aca gga gaa atc aat ggt tct 100 Leu Phe CysLeu Arg Ile Lys Val Leu Thr Gly Glu Ile Asn Gly Ser 10 15 20 25 gcc aattat gag atg ttt ata ttt cac aac gga ggt gta caa att tta 148 Ala Asn TyrGlu Met Phe Ile Phe His Asn Gly Gly Val Gln Ile Leu 30 35 40 tgc aaa tatcct gac att gtc cag caa ttt aaa atg cag ttg ctg aaa 196 Cys Lys Tyr ProAsp Ile Val Gln Gln Phe Lys Met Gln Leu Leu Lys 45 50 55 ggg ggg caa atactc tgc gat ctc act aag aca aaa gga agt gga aac 244 Gly Gly Gln Ile LeuCys Asp Leu Thr Lys Thr Lys Gly Ser Gly Asn 60 65 70 aca gtg tcc att aagagt ctg aaa ttc tgc cat tct cag tta tcc aac 292 Thr Val Ser Ile Lys SerLeu Lys Phe Cys His Ser Gln Leu Ser Asn 75 80 85 aac agt gtc tct ttt tttcta tac aac ttg gac cat tct cat gcc aac 340 Asn Ser Val Ser Phe Phe LeuTyr Asn Leu Asp His Ser His Ala Asn 90 95 100 105 tat tac ttc tgc aaccta tca att ttt gat cct cct cct ttt aaa gta 388 Tyr Tyr Phe Cys Asn LeuSer Ile Phe Asp Pro Pro Pro Phe Lys Val 110 115 120 act ctt aca gga ggatat ttg cat att tat gaa tca caa ctt tgt tgc 436 Thr Leu Thr Gly Gly TyrLeu His Ile Tyr Glu Ser Gln Leu Cys Cys 125 130 135 cag ctg aag ttc tggtta ccc ata gga tgt gca gcc ttt gtt gta gtc 484 Gln Leu Lys Phe Trp LeuPro Ile Gly Cys Ala Ala Phe Val Val Val 140 145 150 tgc att ttg gga tgcata ctt att tgt tgg ctt aca aaa aag aag tat 532 Cys Ile Leu Gly Cys IleLeu Ile Cys Trp Leu Thr Lys Lys Lys Tyr 155 160 165 tca tcc agt gtg cacgac cct aac ggt gaa tac atg ttc atg aga gca 580 Ser Ser Ser Val His AspPro Asn Gly Glu Tyr Met Phe Met Arg Ala 170 175 180 185 gtg aac aca gccaaa aaa tct aga ctc aca gat gtg acc cta 622 Val Asn Thr Ala Lys Lys SerArg Leu Thr Asp Val Thr Leu 190 195 taatatggaa ctctggcacc caggcatgaagcacgttggc cagttttcct caacttgaag 682 tgcaagattc tcttatttcc gggaccacggagagtctgac ttaactacat acatcttctg 742 ctggtgtttt gttcaatctg gaagaatgactgtatcagtc aatggggatt ttaacagact 802 gccttggtac tgccgagtcc tctcaaaacaaacaccctct tgcaaccagc tttggagaaa 862 gcccagctcc tgtgtgctca ctgggagtggaatccctgtc tccacatctg ctcctagcag 922 tgcatcagcc agtaaaacaa acacatttacaagaaaaatg ttttaaagat gccaggggta 982 ctgaatctgc aaagcaaatg agcagccaaggaccagcatc tgtccgcatt tcactatcat 1042 actacctctt ctttctgtag ggrtgagaattcctctttta atcagtcaag ggagatgctt 1102 caaagctggr gctattttat ttctgagatgttgatgtgaa ctgtacatta gtacatactc 1162 agtactctcc ttcaattgct gaaccccagttgaccatttt accaagactt tagatgcttt 1222 cttgtgccct caattttctt tttaaaaatacttctacatg actgcttgac agcccaacag 1282 ccactctcaa tagagagcta tgtcttacattctttcctct gctgctcaat agttttatat 1342 atctatgcat acatatatac acacatatgtatataaaatt cataatgaat atatttgcct 1402 atattctccc tacaagaata tttttgctccagaaagacat gttcttttct caaattcagt 1462 taaaatggtt tactttgttc aagttagtggtaggaaacat tgcccggaat tgaaagcaaa 1522 tttawwttat tatcctattt tctaccattatctatgtttt catggtgcta ttaattacaa 1582 gtttagttct ttttgtagat catattaaaattgcaaacaa aatcatcttt aatgggccag 1642 cattctcatg gggtagagca gaatattcatttagcctgaa agctgcagtt actataggtt 1702 gctgtcagac tatacccatg gtgcctctgggcttgacagg tcaaaatggt ccccatcagc 1762 ctggagcagc cctccagacc tgggtggaattccagggttg agagactccc ctgagccaga 1822 ggccactagg tattcttgct cccagaggctgaagtcaccc tgggaatcac agtggtctac 1882 ctgcattcat aattccagga tctgtgaagagcacatatgt gtcagggcac aattccctct 1942 cataaaaacc acacagcctg gaaattggccctggcccttc aagatagcct tctttagaat 2002 atgatttggc tagaaagatt cttaaatatgtggaatatga ttattcttag ctggaatatt 2062 ttctctactt cctgtctgca tgcccaaggcttctgaagca gccaatgtcg atgcaacaac 2122 atttgtaact ttaggtaaac tgggattatgttgtagttta acattttgta actgtgtgct 2182 tatagtttac aagtgagacc cgatatgtcattatgcatac ttatattatc ttaagcatgt 2242 gtaatgctgg atgtgtacag tacagtacwtaacttgtaat ttgaatctag tatggtgttc 2302 tgttttcagc tgacttggac aacctgactggctttgcaca ggtgttccct gagttgtttg 2362 caggtttctg tgtgtggggt ggggtatggggaggagaacc ttcatggtgg cccacctggc 2422 ctggttgtcc aagctgtgcc tcgacacatcctcatcccaa gcatgggaca cctcaagatg 2482 aataataatt cacaaaattt ctgtgaaatcaaatccagtt ttaagaggag ccacttatca 2542 aagagatttt aacagtagta agaaggcaaagaataaacat ttgatattca gcaactgaaa 2602 aaaaaaaa 2610 4 2072 DNA Rattusnorvegicus CDS (35)...(634) 4 ctggagggga agagtgcagc tgttcctggc agac atgaag ccc tac ttc tcg tgc 55 Met Lys Pro Tyr Phe Ser Cys 1 5 gtc ttt gtcttc tgc ttc cta atc aaa ctt tta aca gga gaa ctc aat 103 Val Phe Val PheCys Phe Leu Ile Lys Leu Leu Thr Gly Glu Leu Asn 10 15 20 gac ttg gcc aatcac agg atg ttt tcg ttt cac gat gga ggt gta cag 151 Asp Leu Ala Asn HisArg Met Phe Ser Phe His Asp Gly Gly Val Gln 25 30 35 att tct tgt aac taccct gag act gtc cag cag tta aaa atg cag ttg 199 Ile Ser Cys Asn Tyr ProGlu Thr Val Gln Gln Leu Lys Met Gln Leu 40 45 50 55 ttc aaa gac aga gaagtc ctc tgc gac ctc acc aag acc aag gga agc 247 Phe Lys Asp Arg Glu ValLeu Cys Asp Leu Thr Lys Thr Lys Gly Ser 60 65 70 gga aac acc gtg tcc atcaag aat ccg atg tcc tgt cca tat cag ctg 295 Gly Asn Thr Val Ser Ile LysAsn Pro Met Ser Cys Pro Tyr Gln Leu 75 80 85 tcc aac aac agt gtc tct tttttc cta gac aac gca gac agc tcc cag 343 Ser Asn Asn Ser Val Ser Phe PheLeu Asp Asn Ala Asp Ser Ser Gln 90 95 100 ggc agc tac ttt tta tgc agcctg tcg att ttc gac cca ccc cct ttt 391 Gly Ser Tyr Phe Leu Cys Ser LeuSer Ile Phe Asp Pro Pro Pro Phe 105 110 115 caa gaa aag aac ctt agt ggagga tat ttg ctt att tat gaa tcc cag 439 Gln Glu Lys Asn Leu Ser Gly GlyTyr Leu Leu Ile Tyr Glu Ser Gln 120 125 130 135 ctt tgt tgc cag ctg aagctt tgg tta ccc gta ggg tgt gca gct ttt 487 Leu Cys Cys Gln Leu Lys LeuTrp Leu Pro Val Gly Cys Ala Ala Phe 140 145 150 gtg gca gcg ctc ctt tttgga tgc ata ttt atc gtc tgg ttt gca aaa 535 Val Ala Ala Leu Leu Phe GlyCys Ile Phe Ile Val Trp Phe Ala Lys 155 160 165 aag aag tac aga tcc agtgtg cac gac cct aat agc gag tac atg ttc 583 Lys Lys Tyr Arg Ser Ser ValHis Asp Pro Asn Ser Glu Tyr Met Phe 170 175 180 atg gcg gca gtc aac acaaac aaa aag tcc aga ctt gca ggt atg acc 631 Met Ala Ala Val Asn Thr AsnLys Lys Ser Arg Leu Ala Gly Met Thr 185 190 195 tca taatctggaacacgggaacc catggaggaa ctacactgtc tagttcccct 684 Ser 200 gaaacttgaatggagaaagt cttctatttt ctggaccaca gggcatctga cttgattaac 744 tactgatacctccttttggk gttttgtttg tctggatcag tgactatcag tcactcggaa 804 tttcagcagactgccctggg tttgctgagt ccttttaagg caaacccctt cttatagaag 864 acccggctcatatgtattca acaaacagac ctcactggga tacaatcccc tctttctgcg 924 cctgcttctagctatgcacc ggccagcaag acaaacatat ctccagcatt tttacaaaaa 984 tgccagggtatgaatctgta aagtacacag gcagccattg accaccgtct gtcctcgttt 1044 tttcagattctatttttttc catagagatc agcattcctt ctagaatcag acagtagagg 1104 gagatgcttcacaacagaag ctcttatgtt tctgagatgt tgatgaattc atgctttagt 1164 accaccatgttctctaacaa cttctatatt ccagctgatc actgcttcag ggcttagatg 1224 cctgcttttgccttcaagtc tccccttaaa gatactccca caggtctact tggtggcctg 1284 cagccactctgaataggaag tttggtctac aatttccccc ctctgctgct caaaaaaaaa 1344 aattagtagatatgattttc ccatattctc cctgccaaag taattttttc cagcaaagac 1404 atctaaattcagttaatatg gtttactgtg ttgatattag tggcagtaaa catttctcag 1464 aatcaaaagcaaattaattt tgcggtggtg tttttctacc attatcttgg gtttccatgg 1524 tgctattactcacaagttta gctatttttt tatgcatcat attaaagttg caagcaagca 1584 gagcaaccctcggttaatgg gcaaacattc tcctggggta gaatgaattg tctatttagc 1644 ccgaaaactgcagtttctgt gggtggctgc cagactacag ccgtgctttg ctctggcttt 1704 gacaggttgaaatagycccc atgascstgg aacagwactc cagactgtgc tggagtccca 1764 aagttaggagggccatggag cctgggacag gctgctgctt tggtctttag gatctaggaa 1824 raattacagaggggccaaga cagagttccc tcccctagaa actgtgcagc ctggaagtca 1884 gccctggcactttaagatag ccttctttag aacatgagtt agttggtagt attctgacgt 1944 gtaaacagcctatkgttgct cggagctgga ccattttctc cacttccctg tctgcatgcc 2004 taagacttctagagcagcca acgtatatgc aacattaaag aaaaaaaaaa aaaaaaaaaa 2064 aaaaaaaa2072 5 603 DNA Mus musculus CDS (1)...(600) 5 atg aag ccg tac ttc tgccat gtc ttt gtc ttc tgc ttc cta atc aga 48 Met Lys Pro Tyr Phe Cys HisVal Phe Val Phe Cys Phe Leu Ile Arg 1 5 10 15 ctt tta aca gga gaa atcaat ggc tcg gcc gat cat agg atg ttt tca 96 Leu Leu Thr Gly Glu Ile AsnGly Ser Ala Asp His Arg Met Phe Ser 20 25 30 ttt cac aat gga ggt gta cagatt tct tgt aaa tac cct gag act gtc 144 Phe His Asn Gly Gly Val Gln IleSer Cys Lys Tyr Pro Glu Thr Val 35 40 45 cag cag tta aaa atg cga ttg ttcaga gag aga gaa gtc ctc tgc gaa 192 Gln Gln Leu Lys Met Arg Leu Phe ArgGlu Arg Glu Val Leu Cys Glu 50 55 60 ctc acc aag acc aag gga agc gga aatgcg gtg tcc atc aag aat cca 240 Leu Thr Lys Thr Lys Gly Ser Gly Asn AlaVal Ser Ile Lys Asn Pro 65 70 75 80 atg ctc tgt cta tat cat ctg tca aacaac agc gtc tct ttt ttc cta 288 Met Leu Cys Leu Tyr His Leu Ser Asn AsnSer Val Ser Phe Phe Leu 85 90 95 aac aac cca gac agc tcc cag gga agc tattac ttc tgc agc ctg tcc 336 Asn Asn Pro Asp Ser Ser Gln Gly Ser Tyr TyrPhe Cys Ser Leu Ser 100 105 110 att ttt gac cca cct cct ttt caa gaa aggaac ctt agt gga gga tat 384 Ile Phe Asp Pro Pro Pro Phe Gln Glu Arg AsnLeu Ser Gly Gly Tyr 115 120 125 ttg cat att tat gaa tcc cag ctc tgc tgccag ctg aag ctc tgg cta 432 Leu His Ile Tyr Glu Ser Gln Leu Cys Cys GlnLeu Lys Leu Trp Leu 130 135 140 ccc gta ggg ttg cca gct ttc gtt gtg gtactc ctt ttt gga tgc ata 480 Pro Val Gly Leu Pro Ala Phe Val Val Val LeuLeu Phe Gly Cys Ile 145 150 155 160 ctt atc atc tgg ttt tca aaa aag aaatac gga tcc agt gtg cat gac 528 Leu Ile Ile Trp Phe Ser Lys Lys Lys TyrGly Ser Ser Val His Asp 165 170 175 cct aat agt gaa tac atg ttc atg gcggca gtc aac aca aac aaa aag 576 Pro Asn Ser Glu Tyr Met Phe Met Ala AlaVal Asn Thr Asn Lys Lys 180 185 190 tct aga ctt gca ggt gtg acc tca taa603 Ser Arg Leu Ala Gly Val Thr Ser 195 200 6 836 DNA Rattus norvegicusCDS (35)...(682) 6 ctggagggga agagtgcagc tgttcctggc agac atg aag ccc tacttc tcg tgc 55 Met Lys Pro Tyr Phe Ser Cys 1 5 gtc ttt gtc ttc tgc ttccta atc aaa ctt tta aca gga gaa ctc aat 103 Val Phe Val Phe Cys Phe LeuIle Lys Leu Leu Thr Gly Glu Leu Asn 10 15 20 gac ttg gcc aat cac agg atgttt tcg ttt cac gat gga ggt gta cag 151 Asp Leu Ala Asn His Arg Met PheSer Phe His Asp Gly Gly Val Gln 25 30 35 att tct tgt aac tac cct gag actgtc cag cag tta aaa atg cag ttg 199 Ile Ser Cys Asn Tyr Pro Glu Thr ValGln Gln Leu Lys Met Gln Leu 40 45 50 55 ttc aaa gac aga gaa gtc ctc tgcgac ctc acc aag acc aag gga agc 247 Phe Lys Asp Arg Glu Val Leu Cys AspLeu Thr Lys Thr Lys Gly Ser 60 65 70 gga aac acc gtg tcc atc aag aat ccgatg tcc tgt cca tat cag ctg 295 Gly Asn Thr Val Ser Ile Lys Asn Pro MetSer Cys Pro Tyr Gln Leu 75 80 85 tcc aac aac agt gtc tct ttt ttc cta gacaac gca gac agc tcc cag 343 Ser Asn Asn Ser Val Ser Phe Phe Leu Asp AsnAla Asp Ser Ser Gln 90 95 100 ggc agc tac ttt tta tgc agc ctg tcg attttc gac cca ccc cct ttt 391 Gly Ser Tyr Phe Leu Cys Ser Leu Ser Ile PheAsp Pro Pro Pro Phe 105 110 115 caa gaa aag aac ctt agt gga gga tat ttgctt att tat gaa tcc cag 439 Gln Glu Lys Asn Leu Ser Gly Gly Tyr Leu LeuIle Tyr Glu Ser Gln 120 125 130 135 ctt tgt tgc cag ctg aag ctt tgg ttaccc gta ggg tgt gca gct ttt 487 Leu Cys Cys Gln Leu Lys Leu Trp Leu ProVal Gly Cys Ala Ala Phe 140 145 150 gtg gca gcg ctc ctt ttt gga tgc atattt atc gtc tgg ttt gca aaa 535 Val Ala Ala Leu Leu Phe Gly Cys Ile PheIle Val Trp Phe Ala Lys 155 160 165 aag aag tac aga tcc agt gtg cac gaccct aat agc gag tac atg ttc 583 Lys Lys Tyr Arg Ser Ser Val His Asp ProAsn Ser Glu Tyr Met Phe 170 175 180 atg gcg gca gtc aac aca aac aaa aagtcc aga ctt gca ggt aca gca 631 Met Ala Ala Val Asn Thr Asn Lys Lys SerArg Leu Ala Gly Thr Ala 185 190 195 ccc ctt agg gct ttg ggg aga gga gaacac tct tca tgt caa gac cgg 679 Pro Leu Arg Ala Leu Gly Arg Gly Glu HisSer Ser Cys Gln Asp Arg 200 205 210 215 aat taatttgttt atttctattttaaaagaaag acattttttc ccctaaagat 732 Asn aatttttgta tttttatgtgaaagtctgaa tcttcatttt aactcgactt atatactctg 792 tggtatatta aaaataatgtttgtgaaaaa aaaaaaaaaa aaaa 836 7 27 DNA Artificial Sequence primer forPCR 7 ctgctcgaga tgaagcccta cttctcg 27 8 32 DNA Artificial Sequenceprimer for PCR 8 accctacggg taacggatcc ttcagctggc aa 32 9 30 DNAArtificial Sequence primer for PCR 9 taactgtttc tcgagaacat gaagtcaggc 3010 30 DNA Artificial Sequence primer for PCR 10 atcctatggg taacggatccttcagctggc 30 11 35 DNA Artificial Sequence primer for PCR 11 cgtgatattgctgaagagct tggcggcgaa tgggc 35 12 34 DNA Artificial Sequence primer forPCR 12 cattcaagtt tcagggaact agtccatgcg tttc 34 13 200 PRT Rattusnorvegicus 13 Met Lys Pro Tyr Phe Ser Cys Val Phe Val Phe Cys Phe LeuIle Lys 1 5 10 15 Leu Leu Thr Gly Glu Leu Asn Asp Leu Ala Asn His ArgMet Phe Ser 20 25 30 Phe His Asp Gly Gly Val Gln Ile Ser Cys Asn Tyr ProGlu Thr Val 35 40 45 Gln Gln Leu Lys Met Gln Leu Phe Lys Asp Arg Glu ValLeu Cys Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser IleLys Asn Pro 65 70 75 80 Met Ser Cys Pro Tyr Gln Leu Ser Asn Asn Ser ValSer Phe Phe Leu 85 90 95 Asp Asn Ala Asp Ser Ser Gln Gly Ser Tyr Phe LeuCys Ser Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Gln Glu Lys AsnLeu Ser Gly Gly Tyr 115 120 125 Leu Leu Ile Tyr Glu Ser Gln Leu Cys CysGln Leu Lys Leu Trp Leu 130 135 140 Pro Val Gly Cys Ala Ala Phe Val AlaAla Leu Leu Phe Gly Cys Ile 145 150 155 160 Phe Ile Val Trp Phe Ala LysLys Lys Tyr Arg Ser Ser Val His Asp 165 170 175 Pro Asn Ser Glu Tyr MetPhe Met Ala Ala Val Asn Thr Asn Lys Lys 180 185 190 Ser Arg Leu Ala GlyMet Thr Ser 195 200 14 200 PRT Mus musculus 14 Met Lys Pro Tyr Phe CysHis Val Phe Val Phe Cys Phe Leu Ile Arg 1 5 10 15 Leu Leu Thr Gly GluIle Asn Gly Ser Ala Asp His Arg Met Phe Ser 20 25 30 Phe His Asn Gly GlyVal Gln Ile Ser Cys Lys Tyr Pro Glu Thr Val 35 40 45 Gln Gln Leu Lys MetArg Leu Phe Arg Glu Arg Glu Val Leu Cys Glu 50 55 60 Leu Thr Lys Thr LysGly Ser Gly Asn Ala Val Ser Ile Lys Asn Pro 65 70 75 80 Met Leu Cys LeuTyr His Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95 Asn Asn Pro AspSer Ser Gln Gly Ser Tyr Tyr Phe Cys Ser Leu Ser 100 105 110 Ile Phe AspPro Pro Pro Phe Gln Glu Arg Asn Leu Ser Gly Gly Tyr 115 120 125 Leu HisIle Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Leu Trp Leu 130 135 140 ProVal Gly Leu Pro Ala Phe Val Val Val Leu Leu Phe Gly Cys Ile 145 150 155160 Leu Ile Ile Trp Phe Ser Lys Lys Lys Tyr Gly Ser Ser Val His Asp 165170 175 Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys180 185 190 Ser Arg Leu Ala Gly Val Thr Ser 195 200 15 216 PRT Rattusnorvegicus 15 Met Lys Pro Tyr Phe Ser Cys Val Phe Val Phe Cys Phe LeuIle Lys 1 5 10 15 Leu Leu Thr Gly Glu Leu Asn Asp Leu Ala Asn His ArgMet Phe Ser 20 25 30 Phe His Asp Gly Gly Val Gln Ile Ser Cys Asn Tyr ProGlu Thr Val 35 40 45 Gln Gln Leu Lys Met Gln Leu Phe Lys Asp Arg Glu ValLeu Cys Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser IleLys Asn Pro 65 70 75 80 Met Ser Cys Pro Tyr Gln Leu Ser Asn Asn Ser ValSer Phe Phe Leu 85 90 95 Asp Asn Ala Asp Ser Ser Gln Gly Ser Tyr Phe LeuCys Ser Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Gln Glu Lys AsnLeu Ser Gly Gly Tyr 115 120 125 Leu Leu Ile Tyr Glu Ser Gln Leu Cys CysGln Leu Lys Leu Trp Leu 130 135 140 Pro Val Gly Cys Ala Ala Phe Val AlaAla Leu Leu Phe Gly Cys Ile 145 150 155 160 Phe Ile Val Trp Phe Ala LysLys Lys Tyr Arg Ser Ser Val His Asp 165 170 175 Pro Asn Ser Glu Tyr MetPhe Met Ala Ala Val Asn Thr Asn Lys Lys 180 185 190 Ser Arg Leu Ala GlyThr Ala Pro Leu Arg Ala Leu Gly Arg Gly Glu 195 200 205 His Ser Ser CysGln Asp Arg Asn 210 215 16 200 PRT Artificial Sequence consensussequence 16 Met Lys Pro Tyr Phe Xaa Xaa Val Phe Val Phe Cys Phe Leu IleLys 1 5 10 15 Leu Leu Thr Gly Glu Xaa Asn Xaa Xaa Ala Asn His Arg MetPhe Ser 20 25 30 Phe His Xaa Gly Gly Val Gln Ile Ser Cys Xaa Tyr Pro GluThr Val 35 40 45 Gln Gln Leu Lys Met Gln Leu Phe Lys Xaa Arg Glu Val LeuCys Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile LysAsn Pro 65 70 75 80 Met Xaa Cys Xaa Tyr Gln Leu Ser Asn Asn Ser Val SerPhe Phe Leu 85 90 95 Xaa Asn Xaa Asp Ser Ser Gln Gly Ser Tyr Xaa Xaa CysSer Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Gln Glu Xaa Asn LeuSer Gly Gly Tyr 115 120 125 Leu Xaa Ile Tyr Glu Ser Gln Leu Cys Cys GlnLeu Lys Leu Trp Leu 130 135 140 Pro Val Gly Cys Ala Ala Phe Val Xaa XaaLeu Leu Phe Gly Cys Ile 145 150 155 160 Xaa Ile Xaa Trp Phe Xaa Lys LysLys Tyr Xaa Ser Ser Val His Asp 165 170 175 Pro Asn Ser Glu Tyr Met PheMet Ala Ala Val Asn Thr Asn Lys Lys 180 185 190 Ser Arg Leu Ala Gly XaaThr Xaa 195 200 17 214 PRT Artificial Sequence consensus sequence 17 MetLeu Xaa Leu Xaa Leu Ala Trp Xaa Leu Xaa Leu Phe Xaa Leu Xaa 1 5 10 15Ile Xaa Val Xaa Xaa Xaa Xaa Ile Xaa Val Xaa Gln Xaa Xaa Xaa Xaa 20 25 30Xaa Ala Xaa Xaa Asn Gly Xaa Xaa Xaa Xaa Xaa Cys Lys Tyr Xaa Xaa 35 40 45Pro Xaa Xaa Xaa Xaa Glu Phe Arg Xaa Xaa Leu Leu Lys Gly Xaa Asp 50 55 60Ser Xaa Val Xaa Xaa Cys Xaa Xaa Xaa Xaa Thr Tyr Xaa Xaa Gly Asn 65 70 7580 Xaa Val Xaa Xaa Lys Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Leu Ser Asn 85 9095 Asn Ser Val Xaa Phe Xaa Leu Gln Asn Leu Xaa Xaa Xaa Xaa Thr Xaa 100105 110 Xaa Tyr Phe Cys Lys Xaa Glu Xaa Met Tyr Pro Pro Pro Tyr Xaa Xaa115 120 125 Xaa Xaa Xaa Asn Gly Thr Xaa Ile His Val Xaa Xaa Xaa Xaa LeuCys 130 135 140 Pro Xaa Xaa Xaa Phe Xaa Xaa Trp Xaa Leu Xaa Xaa Val XaaXaa Xaa 145 150 155 160 Leu Xaa Xaa Tyr Ser Xaa Leu Xaa Thr Ala Xaa IleXaa Xaa Xaa Xaa 165 170 175 Xaa Lys Lys Arg Ser Xaa Leu Xaa Xaa Gly XaaTyr Met Xaa Met Xaa 180 185 190 Pro Xaa Xaa Pro Xaa Xaa Xaa Xaa Lys XaaXaa Gln Pro Tyr Xaa Xaa 195 200 205 Asp Phe Xaa Xaa Xaa Xaa 210 18 6 PRTHomo sapiens 18 Met Tyr Pro Pro Pro Tyr 1 5 19 4 PRT Homo sapiens 19 TyrMet Asn Met 1 20 4 PRT Homo sapiens 20 Tyr Val Lys Met 1 21 6 PRT Homosapiens 21 Phe Asp Pro Pro Pro Phe 1 5 22 4 PRT Homo sapiens 22 Tyr MetPhe Met 1 23 216 PRT Artificial Sequence consensus sequence 23 Met LysPro Tyr Phe Ser Cys Val Phe Val Phe Cys Phe Leu Ile Lys 1 5 10 15 LeuLeu Thr Gly Glu Leu Asn Asp Leu Ala Asn His Arg Met Phe Ser 20 25 30 PheHis Asp Gly Gly Val Gln Ile Ser Cys Asn Tyr Pro Glu Thr Val 35 40 45 GlnGln Leu Lys Met Gln Leu Phe Lys Asp Arg Glu Val Leu Cys Asp 50 55 60 LeuThr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Asn Pro 65 70 75 80Met Ser Cys Pro Tyr Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95Asp Asn Ala Asp Ser Ser Gln Gly Ser Tyr Phe Leu Cys Ser Leu Ser 100 105110 Ile Phe Asp Pro Pro Pro Phe Gln Glu Lys Asn Leu Ser Gly Gly Tyr 115120 125 Leu Leu Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Leu Trp Leu130 135 140 Pro Val Gly Cys Ala Ala Phe Val Ala Ala Leu Leu Phe Gly CysIle 145 150 155 160 Phe Ile Val Trp Phe Ala Lys Lys Lys Tyr Arg Ser SerVal His Asp 165 170 175 Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Val AsnThr Asn Lys Lys 180 185 190 Ser Arg Leu Ala Gly Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 21524 16 PRT Rattus norvegicus 24 Leu Arg Ala Leu Gly Arg Gly Glu His SerSer Cys Gln Asp Arg Asn 1 5 10 15 25 220 PRT Homo sapiens 25 Met Leu ArgLeu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr GlyAsn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp AsnAla Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg GluPhe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val CysVal Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 LysThr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 PheTyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly145 150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala PheIle Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His SerAsp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg LysHis Tyr Gln Pro 195 200 205 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr ArgSer 210 215 220 26 223 PRT Homo sapiens 26 Met Ala Cys Leu Gly Phe GlnArg His Lys Ala Gln Leu Asn Leu Ala 1 5 10 15 Ala Arg Thr Trp Pro CysThr Leu Leu Phe Phe Leu Leu Phe Ile Pro 20 25 30 Val Phe Cys Lys Ala MetHis Val Ala Gln Pro Ala Val Val Leu Ala 35 40 45 Ser Ser Arg Gly Ile AlaSer Phe Val Cys Glu Tyr Ala Ser Pro Gly 50 55 60 Lys Ala Tyr Glu Val ArgVal Thr Val Leu Arg Gln Ala Asp Ser Gln 65 70 75 80 Val Thr Glu Val CysAla Ala Thr Tyr Met Thr Gly Asn Glu Leu Thr 85 90 95 Phe Leu Asp Asp SerIle Cys Thr Gly Thr Ser Ser Gly Asn Gln Val 100 105 110 Asn Leu Thr IleGln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile 115 120 125 Cys Lys ValGlu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly 130 135 140 Asn GlyThr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser 145 150 155 160Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe 165 170175 Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys 180185 190 Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu195 200 205 Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn210 215 220

What is claimed is:
 1. A purified antibody that binds to a polypeptideconsisting of SEQ ID NO:2, wherein the antibody is a human, mouse, rat,guinea pig, rabbit, dog, cat, pig, goat, horse or cow antibody.
 2. Theantibody of claim 1, wherein the antibody is monoclonal.
 3. The antibodyof claim 1, wherein the antibody is polyclonal.
 4. The antibody of claim1, wherein the antibody binds to the extracellular region of thepolypeptide.
 5. The antibody of claim 1, wherein the antibody is ahuman, mouse or rat antibody.
 6. The antibody of claim 1, wherein theantibody is chimeric.
 7. The antibody of claim 1, wherein the antibodyis humanized.
 8. The antibody of claim 1, wherein the antibody is ahuman antibody.
 9. The antibody of claim 2, wherein the antibody bindsto the extracellular region of the polypeptide.
 10. The antibody ofclaim 2, wherein the antibody is a human, mouse or rat antibody.
 11. Theantibody of claim 2, wherein the antibody is chimeric.
 12. The antibodyof claim 2, wherein the antibody is humanized.
 13. The antibody of claim2, wherein the antibody is a human antibody.
 14. A pharmaceuticalcomposition comprising the antibody of claim 1 and a pharmaceuticallyacceptable carrier.
 15. A pharmaceutical composition comprising theantibody of claim 2 and a pharmaceutically acceptable carrier.
 16. Apharmaceutical composition comprising the antibody of claim 3 and apharmaceutically acceptable carrier.
 17. A pharmaceutical compositioncomprising the antibody of claim 4 and a pharmaceutically acceptablecarrier.
 18. A pharmaceutical composition comprising the antibody ofclaim 5 and a pharmaceutically acceptable carrier.
 19. A pharmaceuticalcomposition comprising the antibody of claim 6 and a pharmaceuticallyacceptable carrier.
 20. A pharmaceutical composition comprising theantibody of claim 7 and a pharmaceutically acceptable carrier.
 21. Apharmaceutical composition comprising the antibody of claim 8 and apharmaceutically acceptable carrier.
 22. A pharmaceutical compositioncomprising the antibody of claim 9 and a pharmaceutically acceptablecarrier.
 23. A pharmaceutical composition comprising the antibody ofclaim 10 and a pharmaceutically acceptable carrier.
 24. A pharmaceuticalcomposition comprising the antibody of claim 11 and a pharmaceuticallyacceptable carrier.
 25. A pharmaceutical composition comprising theantibody of claim 12 and a pharmaceutically acceptable carrier.
 26. Apharmaceutical composition comprising the antibody of claim 13 and apharmaceutically acceptable carrier.
 27. A method of inhibitingactivation of lymphocytes in a subject, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 14. 28. A method of inhibiting activation oflymphocytes in a subject, the method comprising administering to thesubject an effective amount of the pharmaceutical composition of claim15.
 29. A method of inhibiting activation of lymphocytes in a subject,the method comprising administering to the subject an effective amountof the pharmaceutical composition of claim
 16. 30. A method ofinhibiting activation of lymphocytes in a subject, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 17. 31. A method of inhibiting activation oflymphocytes in a subject, the method comprising administering to thesubject an effective amount of the pharmaceutical composition of claim18.
 32. A method of inhibiting activation of lymphocytes in a subject,the method comprising administering to the subject an effective amountof the pharmaceutical composition of claim
 19. 33. A method ofinhibiting activation of lymphocytes in a subject, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 20. 34. A method of inhibiting activation oflymphocytes in a subject, the method comprising administering to thesubject an effective amount of the pharmaceutical composition of claim21.
 35. A method of inhibiting activation of lymphocytes in a subject,the method comprising administering to the subject an effective amountof the pharmaceutical composition of claim
 22. 36. A method ofinhibiting activation of lymphocytes in a subject, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 23. 37. A method of inhibiting activation oflymphocytes in a subject, the method comprising administering to thesubject an effective amount of the pharmaceutical composition of claim24.
 38. A method of inhibiting activation of lymphocytes in a subject,the method comprising administering to the subject an effective amountof the pharmaceutical composition of claim
 25. 39. A method ofinhibiting activation of lymphocytes in a subject, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 26. 40. A method of treating glomerulonephritis ina subject, the method comprising administering to the subject aneffective amount of the pharmaceutical composition of claim
 14. 41. Amethod of treating glomerulonephritis in a subject, the methodcomprising administering to the subject an effective amount of thepharmaceutical composition of claim
 15. 42. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 16. 43. A method of treating glomerulonephritis in a subject, themethod comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 17. 44. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 18. 45. A method of treating glomerulonephritis in a subject, themethod comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 19. 46. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 20. 47. A method of treating glomerulonephritis in a subject, themethod comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 21. 48. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 22. 49. A method of treating glomerulonephritis in a subject, themethod comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 23. 50. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 24. 51. A method of treating glomerulonephritis in a subject, themethod comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 25. 52. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 26. 53. An isolated cell that produces the antibody of claim 2.54. An isolated cell that produces the antibody of claim
 4. 55. Anisolated cell that produces the antibody of claim
 5. 56. An isolatedcell that produces the antibody of claim
 6. 57. An isolated cell thatproduces the antibody of claim
 7. 58. An isolated cell that produces theantibody of claim
 8. 59. An isolated cell that produces the antibody ofclaim
 9. 60. An isolated cell that produces the antibody of claim 10.61. An isolated cell that produces the antibody of claim
 11. 62. Anisolated cell that produces the antibody of claim
 12. 63. An isolatedcell that produces the antibody of claim
 13. 64. A purified chimeric,humanized or human monoclonal antibody that binds to a polypeptideconsisting of SEQ ID NO:2.
 65. The antibody of claim 64, wherein theantibody binds to the extracellular region of the polypeptide.
 66. Theantibody of claim 64, wherein the antibody is chimeric.
 67. The antibodyof claim 64, wherein the antibody is humanized.
 68. The antibody ofclaim 64, wherein the antibody is a human antibody.
 69. A pharmaceuticalcomposition comprising the antibody of claim 64 and a pharmaceuticallyacceptable carrier.
 70. A pharmaceutical composition comprising theantibody of claim 65 and a pharmaceutically acceptable carrier.
 71. Apharmaceutical composition comprising the antibody of claim 66 and apharmaceutically acceptable carrier.
 72. A pharmaceutical compositioncomprising the antibody of claim 67 and a pharmaceutically acceptablecarrier.
 73. A pharmaceutical composition comprising the antibody ofclaim 68 and a pharmaceutically acceptable carrier.
 74. A method ofinhibiting activation of lymphocytes in a subject, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 69. 75. A method of inhibiting activation oflymphocytes in a subject, the method comprising administering to thesubject an effective amount of the pharmaceutical composition of claim70.
 76. A method of inhibiting activation of lymphocytes in a subject,the method comprising administering to the subject an effective amountof the pharmaceutical composition of claim
 71. 77. A method ofinhibiting activation of lymphocytes in a subject, the method comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 72. 78. A method of inhibiting activation oflymphocytes in a subject, the method comprising administering to thesubject an effective amount of the pharmaceutical composition of claim73.
 79. A method of treating glomerulonephritis in a subject, the methodcomprising administering to the subject an effective amount of thepharmaceutical composition of claim
 69. 80. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 70. 81. A method of treating glomerulonephritis in a subject, themethod comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 71. 82. A method of treatingglomerulonephritis in a subject, the method comprising administering tothe subject an effective amount of the pharmaceutical composition ofclaim
 72. 83. A method of treating glomerulonephritis in a subject, themethod comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 73. 84. An isolated cell thatproduces the antibody of claim
 64. 85. An isolated cell that producesthe antibody of claim
 65. 86. An isolated cell that produces theantibody of claim
 66. 87. An isolated cell that produces the antibody ofclaim
 67. 88. An isolated cell that produces the antibody of claim 68.89. A purified chimeric, humanized or human monoclonal antibody thatbinds to a polypeptide consisting of SEQ ID NO:13.
 90. The antibody ofclaim 89, wherein the antibody binds to the extracellular region of thepolypeptide.
 91. The antibody of claim 89, wherein the antibody ischimeric.
 92. The antibody of claim 89, wherein the antibody ishumanized.
 93. The antibody of claim 89, wherein the antibody is a humanantibody.
 94. A pharmaceutical composition comprising the antibody ofclaim 89 and a pharmaceutically acceptable carrier.
 95. A pharmaceuticalcomposition comprising the antibody of claim 90 and a pharmaceuticallyacceptable carrier.
 96. A pharmaceutical composition comprising theantibody of claim 91 and a pharmaceutically acceptable carrier.
 97. Apharmaceutical composition comprising the antibody of claim 92 and apharmaceutically acceptable carrier.
 98. A pharmaceutical compositioncomprising the antibody of claim 93 and a pharmaceutically acceptablecarrier.
 99. An isolated cell that produces the antibody of claim 89.100. An isolated cell that produces the antibody of claim
 90. 101. Anisolated cell that produces the antibody of claim
 91. 102. An isolatedcell that produces the antibody of claim
 92. 103. An isolated cell thatproduces the antibody of claim
 93. 104. A purified antibody that bindsto a polypeptide consisting of SEQ ID NO:14, wherein the antibody is ahuman, rat, guinea pig, rabbit, dog, cat, pig, goat, horse or cowantibody.
 105. The antibody of claim 104, wherein the antibody ismonoclonal.
 106. The antibody of claim 104, wherein the antibody ispolyclonal.
 107. The antibody of claim 104, wherein the antibody bindsto the extracellular region of the polypeptide.
 108. The antibody ofclaim 104, wherein the antibody is a human, rat or guinea pig antibody.109. The antibody of claim 104, wherein the antibody is chimeric. 110.The antibody of claim 104, wherein the antibody is humanized.
 111. Theantibody of claim 104, wherein the antibody is a human antibody. 112.The antibody of claim 105, wherein the antibody binds to theextracellular region of the polypeptide.
 113. The antibody of claim 105,wherein the antibody is a human, rat or guinea pig antibody.
 114. Theantibody of claim 105, wherein the antibody is chimeric.
 115. Theantibody of claim 105, wherein the antibody is humanized.
 116. Theantibody of claim 105, wherein the antibody is a human antibody.
 117. Apharmaceutical composition comprising the antibody of claim 104 and apharmaceutically acceptable carrier.
 118. A pharmaceutical compositioncomprising the antibody of claim 105 and a pharmaceutically acceptablecarrier.
 119. A pharmaceutical composition comprising the antibody ofclaim 106 and a pharmaceutically acceptable carrier.
 120. Apharmaceutical composition comprising the antibody of claim 107 and apharmaceutically acceptable carrier.
 121. A pharmaceutical compositioncomprising the antibody of claim 108 and a pharmaceutically acceptablecarrier.
 122. A pharmaceutical composition comprising the antibody ofclaim 109 and a pharmaceutically acceptable carrier.
 123. Apharmaceutical composition comprising the antibody of claim 110 and apharmaceutically acceptable carrier.
 124. A pharmaceutical compositioncomprising the antibody of claim 111 and a pharmaceutically acceptablecarrier.
 125. A pharmaceutical composition comprising the antibody ofclaim 112 and a pharmaceutically acceptable carrier.
 126. Apharmaceutical composition comprising the antibody of claim 113 and apharmaceutically acceptable carrier.
 127. A pharmaceutical compositioncomprising the antibody of claim 114 and a pharmaceutically acceptablecarrier.
 128. A pharmaceutical composition comprising the antibody ofclaim 115 and a pharmaceutically acceptable carrier.
 129. Apharmaceutical composition comprising the antibody of claim 116 and apharmaceutically acceptable carrier.
 130. An isolated cell that producesthe antibody of claim
 105. 131. An isolated cell that produces theantibody of claim
 107. 132. An isolated cell that produces the antibodyof claim
 108. 133. An isolated cell that produces the antibody of claim109.
 134. An isolated cell that produces the antibody of claim
 110. 135.An isolated cell that produces the antibody of claim
 111. 136. Anisolated cell that produces the antibody of claim
 112. 137. An isolatedcell that produces the antibody of claim
 113. 138. An isolated cell thatproduces the antibody of claim
 114. 139. An isolated cell that producesthe antibody of claim
 115. 140. An isolated cell that produces theantibody of claim
 116. 141. A purified chimeric, humanized or humanmonoclonal antibody that binds to a polypeptide consisting of SEQ IDNO:14.
 142. The antibody of claim 141, wherein the antibody binds to theextracellular region of the polypeptide.
 143. The antibody of claim 141,wherein the antibody is chimeric.
 144. The antibody of claim 141,wherein the antibody is humanized.
 145. The antibody of claim 141,wherein the antibody is a human antibody.
 146. A pharmaceuticalcomposition comprising the antibody of claim 141 and a pharmaceuticallyacceptable carrier.
 147. A pharmaceutical composition comprising theantibody of claim 142 and a pharmaceutically acceptable carrier.
 148. Apharmaceutical composition comprising the antibody of claim 143 and apharmaceutically acceptable carrier.
 149. A pharmaceutical compositioncomprising the antibody of claim 144 and a pharmaceutically acceptablecarrier.
 150. A pharmaceutical composition comprising the antibody ofclaim 145 and a pharmaceutically acceptable carrier.
 151. An isolatedcell that produces the antibody of claim
 141. 152. An isolated cell thatproduces the antibody of claim
 142. 153. An isolated cell that producesthe antibody of claim
 143. 154. An isolated cell that produces theantibody of claim
 144. 155. An isolated cell that produces the antibodyof claim 145.